Novel shigella protein antigens and methods

ABSTRACT

The present invention relates to protein antigens IcsP2 and SigA2 from  Shigella  that are common among numerous  Shigella  types and species and which can protect against shigellosis or other enteric infections when administered as vaccines. In addition, the present invention relates to antigens that are in common between  Shigella  species and enteroinvasive  Escherichia coli  (EIEC). The invention also relates to the use of antibodies raised against these antigens and of DNA probes for use in the diagnosis of  Shigella  and EIEC infections.

CROSS REFERENCE TO PRIOR APPLICATIONS

This application claims priority pursuant to 35 U.S.C. §119 to U.S.Provisional Patent Application No. 61/107,306, filed Oct. 21, 2008,which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel protein antigens of Shigellaspp., and also present on Escherichia coli enteroinvasive strains andmethods for use of these antigens for developing vaccines againstshigellosis caused by Shigella species and serotypes, and againstdisease caused by enteroinvasive E. coli (EIEC) bacteria. The inventionalso relates to use of the novel antigens and corresponding antibodiesfor diagnosis of shigellosis and for identification of Shigellabacteria.

BACKGROUND OF THE INVENTION

Shigella spp. is a Gram-negative bacterial pathogen that causesbacillary dysentery in humans by infecting epithelial cells of thecolon. Shigella primarily infects intestinal epithelial cells (TECs).Shigella expresses several proteins that provide a mechanism fordelivering effectors that induce bacterial uptake into the host cell viaphagocytosis. To accomplish the injection of the effectors, Shigella usea type III secretion (TTS) system to induce their entry into epithelialcells and to trigger apoptosis in infected macrophages.

Bacteria of Shigella spp., including S. dysenteriae, S. flexneri, S.boydii and S. sonnei, are responsible for shigellosis in humans, adisease characterized by the destruction of the colonic epithelium thatis responsible for 1 million deaths per year, mostly children indeveloping countries.

There are 15 serotypes in S. dysenteriae, 14 serotypes and subtypes arerecognized in S. flexneri, S. boydii has 20 serotypes and a singleserotype exist within S. sonnei although their prevalence is not evenlydistributed. The most prevalent Shigella spp. in industrializedcountries and of increasing prevalence in some Latin American countriesis S. sonnei. S. dysenteriae type 1, which can produce Shiga-toxin, cancause high morbidity and mortality. S. flexneri is most prevalent inendemic region of developing countries. The World Health Organization(WHO) considered the development of a vaccine against shigellosis apriority for developing countries.

Although control and treatment of shigellosis outbreaks with antibioticsis possible, the high cost of antibiotics and the constant emergence ofantibiotic resistant Shigella species, even to the newest antibiotics,underscores the need for an effective vaccine to help control Shigellaand related enteroinvasive E. coli diseases in the developing regions ofthe world (12).

To establish a successful infection, Shigella finely regulates thehost's immune response, especially those responses leading toinflammation. In contrast to Salmonella Typhimurium, Shigella isinefficient at invading the apical pole of polarized intestinalepithelial cells. Instead, Shigella requires transmigration ofpolymorphonuclear leucocytes (PMN) to disrupt the epithelial barrier,facilitating cell invasion via the basolateral pole of epithelial cells(26). The host's inflammatory response, facilitated by cells of theinnate immune system, attracts PMN to the site of inflammation.Therefore, triggering inflammation at the early stage of infection isrequired for cell invasion by Shigella. Bacteria that reach theintracellular compartment of the cells grow and spread from cell tocell, protected from host immune defenses. But, infected epithelialcells play a large role in the inflammatory process, both as sentinelsthat detect bacterial invasion and as a major source of mediators,particularly cytokines and chemokines that initiate and orchestratemucosal inflammation. Recognition of the bacteria by the epithelialcells occurs essentially intracellularly via a cytoplasmic molecule,Nodl/CARD4 that senses a microbial motif, the peptidoglycan (8). Nod 1activation induces other proinflammatory signaling pathways includingNF-κB and c-Jun N-terminal kinase (JNK) that lead to the expression ofchemokines, such as interleukin 8 (IL-8). Thus triggering excessiveinflammation is detrimental to Shigella's survival in the host.

Natural Shigella infections confer immunity and provide protectionagainst subsequent infection with homologous virulent Shigella (5). Thisexclusively human disease is transmitted directly via the fecal-oralroute from an infected patient or indirectly through contaminated foodand water. It is a highly contagious infection, capable of transmissionwith as few as 100 microorganisms (6). Epidemiologic and volunteerstudies have revealed that protective immunity against Shigella isdirected against the LPS or O-specific antigen and is therefore relatedto serotype. Many approaches have been used for Shigella vaccinesincluding use of live attenuated Shigella (16, 22), killed Shigellawhole bacteria (18), and Shigella lipopolyssacharide (LPS) orO-polysaccharides conjugated to carriers such as proteosomes (24),tetanus toxoid (25) and ribosomes (31). Despite many years of extensiveresearch, an effective and inexpensive vaccine against these Shigellaspecies is not yet available.

The use of attenuated strains of Shigella as live oral vaccines has beendemonstrated to induce protective efficacy. Results from the clinicaltrials of genetically well characterized, invasive Shigella vaccines arepromising. CVD1208, SC602, WRSS1 add WRSd1 vaccine candidates,administered orally, are safe and immunogenic in volunteer trials and,in the case of SC602, have been demonstrated to protect againstdysentery (11, 16, 17, 33). Clinical trials with CVD1208 demOnstratedthat the symptoms of mild fever and diarrhea, which are seen with someof the live Shigella vaccines, can be reduced by elimination of the senand set genes from the vaccine strain. Duplication of a successfulstrategy in one serotype to other serotypes is an ongoing area ofresearch but will eventually require use of a polyvalent mixture ofShigella strains of different serotypes that can protect against most ofthe Shigella (21). A recent multicentre study of Shigella diarrhoea insix Asian countries indicated that the relative distribution of Shigellaspecies isolated from patients varied from different countries andsites. Moreover, S. flexneri serotypes were highly heterogeneous intheir distribution from site to site, and even from year to year. Theheterogeneous distribution of Shigella species and serotypes suggestthat multivalent or cross-protective Shigella vaccines will be needed toprevent shigellosis worldwide (35). A vaccine that aims to conferbroad-spectrum coverage would require inclusion of all of the importantShigella serotypes (21). To resolve this dilemma a vaccine strategybased on the use of ‘pentavalent formulations’, comprising S. flexneri2a, 3a and 6 strains along with the attenuated S. sonnei and S.dysenteriae 1 strains has been advocated (Noriega et al, 1994).Alternatively, use of complex structures comprised of serotype-specificand cross-reactive antigens from Shigella, such as whole bacteria eitherkilled or live-attenuated, could be considered as an approach tovaccinate against infections caused by the most common species andserotypes of Shigella. Intranasally administered Invaplex, a purifiedcomplex from Shigella water extract composed of the Ipa proteins andLPS, has been proposed and is in Phase 1 trials currently at the WalterReed Army Institute of Research (WRAIR) (23).

There is thus a need for an effective vaccine to help control Shigellaand related enteroinvasive E. coli diseases in the developing regions ofthe world.

SUMMARY OF THE INVENTION

In certain embodiments, the invention relates to a vaccine compositionfor immunizing a mammal against Shigella comprising an amount of aShigella from the group consisting of IcsP2 and SigA2 proteins effectiveto elicit an immune response against Shigella, and a pharmaceuticallyacceptable carrier or diluent. In certain embodiments, the ShigellaIcsP2 and SigA2 proteins are chemically conjugated or genetically fusedwith other proteins.

In additional embodiments, the invention relates to a vaccinecomposition for immunizing a mammal against Shigella comprising anamount of Shigella IcsP2 effective to elicit an immune response againstShigella, and a pharmaceutically acceptable carrier or diluent.

In yet additional embodiments, the invention relates to a vaccinecomposition for immunizing a mammal against Shigella comprising anamount of Shigella SigA2 effective to elicit an immune response againstShigella, and a pharmaceutically acceptable carrier or diluent.

In yet additional embodiments, the invention relates to a vaccinecomposition for immunizing a mammal against Shigella comprising anamount of chemically conjugated or genetically fused IcsP2 and SigA2.

In certain embodiments, the vaccine composition further comprises anadjuvant.

In certain embodiments, the adjuvant is an oil phase of an emulsionselected from a group consisting of a water-in-oil emulsion and a doubleoil emulsion.

In additional embodiments, the invention relates to an isolatedpolypeptide comprising an amino acid sequence as set forth in SEQ IDNO:2 (for Shigella IcsP2).

In additional embodiments, the invention relates to an isolatedpolypeptide comprising an amino acid sequence as set forth in SEQ ID NO:4 (for SigA2).

In additional embodiments, the invention relates to a an isolatednucleic acid sequence encoding the Shigella IcsP2 polypeptide.

In additional embodiments, the invention relates to an isolated nucleicacid sequence encoding the SigA2 polypeptide.

In additional embodiments, the invention relates to an isolated nucleicacid sequence encoding the Shigella IcsP2 polypeptide and the SigA2polypeptide.

In additional embodiments, the invention relates to a vector comprisingthe nucleic acid sequence of claim 8, 9, or 10. In additionalembodiments, the invention relates to a host cell transfected with anyof the vectors described herein.

In additional embodiments, the invention relates to a method forproducing Shigella IcsP2 and SigA2 polypeptides comprising culturing anyof the host cells described herein under suitable conditions for proteinexpression and collecting said polypeptides from the cultured cells.

In additional embodiments, the invention relates to an immunogeniccomposition comprising a) Shigella IcsP2 and SigA2, and b) an adjuvant,wherein the amounts of a) and b) in combination are effective to elicitan immune response against Shigella.

In additional embodiments, the invention relates to a method of treatinga mammal suffering from or susceptible to a pathogenic infection,comprising administering an effective amount of the vaccine compositiondescribed herein. In certain embodiments, the effective amount of thevaccine composition ranges between about 10 micrograms to about 2milligrams

In additional embodiments, the invention relates to a method formodulating the immune response of a mammal comprising administering aneffective amount of any one of the vaccine compositions describedherein. In certain embodiments, the effective amount of the vaccinecomposition is from about 10 micrograms to about 2 milligrams.

In additional embodiments, the invention relates to an antibody thatspecifically binds to the Shigella IcsP2 polypeptide.

In additional embodiments, the invention relates to an antibody thatspecifically binds to the SigA2 polypeptide. In certain embodiments, theantibody further comprises a label. In additional embodiments, the labelis selected from the group consisting of an enzyme, protein, peptide,antigen, antibody, lectin, carbohydrate, biotin, avidin, radioisotope,toxin and heavy metal. In additional embodiments, the antibody is ahumanized antibody. In additional embodiments, the antibody is aCDR-grafted antibody. In additional embodiments, the antibody is achimeric antibody. In additional embodiments, the antibody is anantibody fragment. In additional embodiments, the antibody is amonoclonal antibody. In additional embodiments, the antibody is apolyclonal antibody.

In additional embodiments, the invention relates to a conjugate moleculecomprising a saccharide comprising an O antigen of Shigella bacteriacovalently bound to a Shigella IcsP2 or SigA2 protein.

In additional embodiments, the invention relates to a conjugate moleculecomprising a saccharide comprising an O antigen of Shigella bacteriacovalently bound to a polypeptide as described herein.

In additional embodiments, the invention relates to a vaccine comprisinga conjugate molecule as described herein for immunizing a mammal againstshigellosis.

In additional embodiments, the invention relates to a conjugate moleculecomprising a saccharide from a bacteria non related to the genusShigella, said saccharide being covalently bound to a Shigella IcsP2 orSigA2 protein.

In additional embodiments, the invention relates to a conjugate moleculecomprising a saccharide from a bacteria non related to the genusShigella, said saccharide being covalently bound to a polypeptide asdescribed herein.

In additional embodiments, the invention relates to a vaccine comprisinga conjugate molecule as described herein for immunizing a mammal againstshigellosis and typhoid disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that immunization with IcsP2 protein protected animalsagainst lung challenge with S. flexneri 2a. The results also show thatintranasal administration of a live-attenuated S. flexneri vaccinestrain (SC602) protected mice against challenge.

FIG. 2 shows mucosal immunization with IcsP2 protected mice againstpneumonia induced by distinct serotypes of Shigella flexneri

FIG. 3 shows that mucosal immunization with IcsP2 protected mice againstpneumonia induced by Shigella dysenteriae type 1.

FIG. 4 shows that mucosal immunization with SigA2 protected against S.flexneri 2a (2457T) challenge but not against S. flexneri 5a (M90T).

FIG. 5 shows that mucosal administration of SigA2 or IcsP2 giventogether with CT adjuvant induced serum antibody responses.

FIG. 6A-B are graphs illustrating that animals immunized with SigA2mounted predominantly IgA- and also IgG-ASC responses in both spleen(FIG. 6A) and lungs (FIG. 6B).

FIG. 7 is a graph illustrating that systemic (i.p.) as well as mucosal(i.n.) immunization with SigA2 induced antibody responses in the lungsand in serum.

FIG. 8 shows the recognition of SigA2 protein in Shigella strains bymice antisera raised against SigA2 protein.

FIG. 9 shows test antisera to SigA2 inhibited plaque formation inducedby S. flexneri.

FIG. 10 is photograph of a gel showing the presence of sigA gene andicsP gene in each serotype of Shigella spp.

FIGS. 11A-D show that antibodies against SigA2 inhibitedkeratoconjunctivitis by S. flexneri 2a.

FIG. 12 shows the DNA sequence of the IcsP2 fragment (SEQ ID NOS 1 and 2disclosed as the DNA and corresponding amino acid sequences,respectively) and its position within the sequence of full-length IcsP(SEQ ID NOS 5 and 6 disclosed as the DNA and corresponding amino acidsequences, respectively). The IcsP2 fragment was extracted from thefull-length iscP gene of the whole genomic sequence of strain S.flexneri 2a 2457T.

FIG. 13 shows the DNA sequence of the SigA2 fragment (SEQ ID NOS 3 and 4disclosed as the DNA and corresponding amino acid sequences,respectively) and its position within the sequence of full-length SigA(SEQ ID NOS 7 and 8 disclosed as the DNA and corresponding amino acidsequences, respectively). The SigA2 fragment was extracted from thefull-length sigA gene of the whole genomic sequence of strain S.flexneri 2a 2457T.

DETAILED DESCRIPTION

The present invention relates to the identification of IcsP and SigA ascandidate protein antigens in the composition of vaccines againstShigella infections. More specifically, the present invention relates tospecific polypeptide moieties or fragments of Shigella IcsP and SigA,capable of inducing protective immune responses against infection causedby virulent Shigella bacteria. Said specific polypeptides are referredto hereafter to as IcsP2 and SigA2, respectively. IcsP2 is common to allShigella spp. and is also present on EIEC. SigA2 is present on strainsof S. flexneri 2a and of S. boydii and S. sonnei.

The present invention relates to protein antigens IcsP and SigAidentified as surface-associated and/or secreted from Shigella that arecommon to Shigella types and species including S. flexneri, S. sonnei,S. boydii and S. dysenteriae and which can protect against shigellosisand other enteric infections when administered as vaccines. In addition,the present invention relates to certain of such antigens that are alsocommon between Shigella species and EIEC. The invention also relates tothe use of antibodies raised against these antigens and of DNA probesfor use in the diagnosis of Shigella and EIEC infections.

Antigen Identification and Characteristics of the Antigens.

The whole genome sequences of Shigella spp., S. flexneri 2a strains2457T (37) and Sf301 (15), S. flexneri 5b strain Sf8401 (20), S.dysenteriae 1, strain Sd197, S. boydii serotype 4,strain Sb227 and S.sonnei strain Ss046 are available (38, 39). Besides the whole genomesequences of those Shigella strains, the complete nucleotide sequencesof the virulence plasmid from a number of Shigella spp. are alsoavailable (3, 14).

IcsP

Shigella movement within the host cell cytoplasm is dependent on theability of the bacterium to recruit host cell actin to its surface toform an actin tail, which propels the bacterium from one cell to another(2). Actin tail assembly is mediated by a single bacterial protein IcsA,which is found on the outer surface at one pole of the bacterium, thatpole at which actin assembly occurs (27). The IcsA protein is localizedto the old pole of the bacterium and is both necessary and sufficientfor actin assembly (9). The icsA gene is located on the Shigellavirulence plasmid, and since it is essential for the movement of thebacteria, it is present among all the serotypes and species (38). TheIcsA protein is comprised of two domains: the α-domain (residues 53 to758) contains the determinant for actin assembly and extends from thebacterial surface into the extracellular environment, whereas theβ-domain (residues 759 to 1102) is embedded in the outer membrane (32).

IcsA is slowly cleaved from the bacterial surface by the outer membraneprotease IcsP (7). IcsP is encoded by a monocistronic operon on thelarge (230-kb) virulence plasmid of Shigella (3). Absence of IcsP leadsto an alteration in the distribution of surface IcsA, such that thepolar cap is maintained and some IcsA is distributed along the lateralwalls of the bacterium. The amino acid sequence of 327 a.a polypeptidehas 58% sequence identity to each of E. coli proteases OmpP and OmpT,42% sequence identity to the Salmonella Typhimurium protease Eprecursor, PrtA and 40% sequence identity to the Yersinia pestisfibrinolysin precursor Pla (29).

SigA

The sigA gene is situated on the pathogenicity island of Shigellaflexneri 2a chromosomal DNA. Although sigA was believed to beexclusively found in serotype 2a of S. flexneri, the presence of sigAhas also been reported in S. boydii and S. sonnei (38). Sequenceanalysis indicates that sigA encodes a 139 lcDa protein which belongs tothe SPATE (serine protease autotransporters of Enterobacteriaceae)subfamily of autotransporter proteins (1). Amino acid identity of SigAprotein as compared to enteroaggregative E. coli autotransporter Pet, anenterotoxic and cytopathic protease, is 58% (1). SigA mutant bacteriashows reduced fluid accumulation as compared to the wild type Shigella,however, the mutant bacteria still is capable of inducing substantialfluid accumulation, which implies that SigA is only one of a number ofenterotoxins produced by S. flexneri 2a.

Identification of novel antigens common to most, if not all, species andserotypes of Shigella would be ideal provided that such antigens areindeed protective. Genomics and proteomics have had a dramatic effect onthe ability to find new vaccine targets and develop effective vaccinesand some Shigella surface proteins are recognized as possible vaccinecandidates for Shigellosis (13, 19, 36).

The present invention also relates to the cloning, expression andpurification of Shigella proteins of interest. The purified proteinswere used to immunize and evaluate protective immunity in three animalmodels 1) mouse pneumonia model, 2) guinea pig keratoconjunctivitismodel, and 3) guinea pig colitis model (such models are described in 10,28, 30).

In certain embodiments, the invention provides methods for screeningcommon protein antigens of Shigella spp and the methods used in thisinvention may be applied to screening of protein antigens of mucosalpathogens.

In certain embodiments, the invention provides methods for producingspecific antibodies to said Shigella IcsP2 and SigA2 polypeptides andfor preparing corresponding DNA probes specific for said commonantigens. Such antibodies and DNA probes can be used for detection ofbacteria expressing said common antigens or corresponding genes, for thediagnosis of bacillary dysentery caused by Shigella and EIEC.Therapeutic antibodies can also be used to treat patients suffering fromacute bacillary dysentery caused by bacteria expressing thecorresponding protein antigens.

Diagnosis of Bacillary Dysentery

Aside from Shigella ssp, certain strains of Escherichia coli can causedysentery. Currently, there are four recognized classes ofenterovirulent E. coli (collectively referred to as the EEC group) thatcause gastroenteritis in humans. E. coli is part of the normalintestinal flora of humans and other primates. A minority of E. colistrains are capable of causing human illness by several differentmechanisms. Among these are the enteroinvasive (EIEC) strains. It isunknown what foods may harbor these pathogenic enteroinvasive (EIEC)strains responsible for a form of bacillary dysentery. Enteroinvasive E.coli (EIEC) may produce an illness known as bacillary dysentery. TheEIEC strains responsible for this syndrome are closely related toShigella ssp. Following the ingestion of EIEC, the organisms invade theepithelial cells of the intestine, resulting in a mild form ofdysentery, often mistaken for dysentery caused by Shigella species. Theillness is characterized by the appearance of blood and mucus in thestools of infected individuals. The diagnosis of Shigella and EIECinfection is relatively difficult since the bacteria must be isolatedfrom stools and the infectious dose of Shigella and EIEC is thought tobe as few as 10 to 100 organisms.

The culturing of the organism from the stools of infected individualsand the demonstration of invasiveness of isolates in tissue culture orin a suitable animal model is necessary to diagnose dysentery caused bythis organism. However, such an approach is cumbersome andtime-consuming.

Echeverria P. et al. describe that “( . . . ) the four Shigella species(S. dysenteriae, S. flexneri, S. boydii, and S. sonnei) are classicallyidentified by culture of fecal specimens on selective media and testingof isolates for agglutination by species-specific antisera. DNA probeshave been used to identify both lactose-fermenting andnon-lactose-fermenting EIEC as well as Shigella isolates that do notagglutinate in antisera. These DNA probes are not necessary for theidentification of Shigella if a competent bacteriology laboratory withShigella antisera is available. The clinical illness associated withEIEC infections is similar to shigellosis. Fewer children with EIECinfections than with shigellosis, however, have occult blood in stool(36% vs. 82%) and more than 10 fecal leukocytes per high-power field(36% vs. 67%). Standard bacteriologic methods and testing of E. coliisolates for hybridization with the Shigella/EIEC probe are currentlythe most sensitive means of diagnosing infections caused by theseenteric pathogens. A more rapid method of identifying Shigella and EIECinfections in a situation where a bacteriology laboratory is notavailable involves immunological assays” (40), Rev. Infect. Dis. 1991March-April; 13 Suppl 4:S220-5).

The present invention relates in part to the finding that IcsP2 is alsoexpressed by EIEC and its sequence is highly homologous to that ofShigella IcsP2, but not to genes of the other 3 classes ofenterovirulent E. coli (EEC). Consequently, an aspect of the presentinvention relates to IcsP2 as a protective agent against EIEC as well asDNA probes and specific antibodies to Shigella IcsP2 that can be used todiagnose dysentery caused by both Shigella and EIEC.

In one embodiment, the Shigella polypeptide antigens disclosed in thepresent invention are administered with a pharmaceutically acceptablediluent. Such formulations can be administered by an injection(subcutaneous, intradermal, intramuscular) or applied topically onto theskin using an adhesive patch. Alternatively, the vaccine is administeredby a mucosal route (oral, buccal, sublingual, nasal drops, aerosol,rectal) using a pharmaceutically acceptable vehicle. The antigens canalso be mixed with an adjuvant to enhance the ensuing immune responses.Example of such adjuvants are without being limited to, aluminium salts,ISCOMs, saponin-based adjuvants, oil-in-water and water-in-oilemulsions, toll-like receptor ligands such as muramyl dipeptide, E. coliLPS, oligonucleotides comprised of unmethylated DNA, poly I:C,lipoteichoic acid, peptidoglycan. Enterotoxins and their adjuvant activederivatives such as cholera toxin, heat-labile E. coli enterotoxin,pertussis toxin, shiga toxin and analogs.

In a further embodiment of the invention the antigens disclosed arecloned and expressed in non virulent or in an attenuated bacteria andthe later are used as vectors containing a DNA promoter element capableof initiating the synthesis of mRNA operably linked to an open readingframe containing one or both of the genes encoding Shigella IcsP andSigA. The resultant protein(s) is(are) exported and assembled on thebacterial surface and/or periplasm. Such non virulent or attenuatedbacteria can then be used as oral or mucosal vaccine. Examples ofbacterial vectors are known in the art, such as E. coli, Salmonellaspp., Shigella spp., Vibrio cholera, Bacillus spp, Clostridium spp,Listerium monocytogenes, Mycobacterium spp., Lactobacillus spp.,Lactococcus spp., Streptococcus gordonii. In another embodiment, theIcsP and the SigA antigens are being overexpressed in non virulentstrains or mutant strains of Shigella that have been equipped with asuitable promoter. Such bacteria expressing either IcsP or SigA antigensor both can then be used as live vaccines against shigellosis.Alternatively, such overexpressing strains can be inactivated withformalin or by heating and the resulting bacteria can be used as killedvaccines. Further embodiments of the invention are vectors used totransform Shigella species which results in the periplasmic expressionof heterologous antigens. This expression is not likely to alter eitherShigella's natural tissue tropism (colonic epithelium) following oraladministration or significantly reduce strain invasiveness. SuitableShigella species include live, attenuated vaccine strains of S. sonnei,S. dysenteriae, Sflextieri, and S. boydii. Exemplified transformedShigella strains include Shigella vaccine strain, e.g. Shigella flexneri2a (SC608(3098)), Shigella flexneri 2a (SC608(cfaAE)), Shigella flexneri2a (SC608(pCFAI)) and Shigella flexneri 2a (SC608(pCFAI/LTB)). Thesestrains are characterized as having deletions in icsA, a gene thatenables intracellular and intercellular spread of Shigella in hostepithelial cells and in the gene iucA that plays a role in ironacquisition by the bacteria. These transformed Shigella strains aresuitable for use in immunogenic composition, in particular oral ormucosally administered vaccines. Other bacteria have been described andare well known in the art for use as vector systems:

-   -   Salmonella and E. coli bacterial surface proteins have been used        as carriers or vehicles of foreign epitopes for various        purposes, including the development of live vaccines (U.S. Pat.        No. 5,348,867, Inventors Georgiou, George, Francisco, Joseph A,        Earhart, Charles F.)    -   Lactobacillus harboring an expression cassette encoding a signal        sequence, wherein the biologically active polypeptide is linked        to a heterologous carboxy-terminal target region.        (WO/2005/012491, PCT/US2004/002460) Inventors: CHANG, Chia-Hwa,        LIU, Xiaowen et al.)    -   Bacterial surface protein expression: Smit, John; and, Nina        Agabian; “Cloning of the Major Protein of the Caulobacter        crescentus Periodic Surface Layer: Detection and        Characterization of the Cloned Peptide by Protein Expression        Assays” (1984) J. Bacteriol. 160, 1137-1145. U.S. Pat. No.        5,500,353.    -   Compartmentalization of recombinant polypeptides in host cells.        (PCT/EP00/00686, U.S. Pat. No. 6,610,517, Inventor Werner        Lubitz).    -   Yeast cell surface display of proteins and uses thereof (U.S.        Pat. No. 6,423,538) Wittrup, K. Dane et al.).    -   Recombinant mycobacteria, particularly recombinant M. bovis BCG,        which express heterologous DNA encoding a product (protein or        polypeptide) of interest (U.S. Pat. No. 5,591,632) O′Donnell,        Michael A. et al.)    -   Use of gram-positive bacteria to express recombinant proteins        (U.S. Pat. No. 5,821,088) Darzins, Aldis et al. Gianni Pozzi et        al., “Delivery and Expression of a Heterologous Antigen on the        Surface of Streptococci”, Infection and Immunity (May 1992)        60:1902-1907    -   Method for expression and secretion in bacillus (U.S. Pat. No.        5,032,510)—S. Kovacevic et al.

The vectors that are used to introduce IcsP and/or SigA polypeptidesinto mammalian cells, tissues, organs or organisms also compriseattenuated viruses equipped with a suitable promoter element thatcontrol expression of the transgenes encoding IcsP and/or SigA can beprepared and used to produce IcsP and/or SigA. Examples of viral vectorsthat can be used to express IcsP and/or SigA include without beinglimited to adenoviruses, polioviruses, a sindbis virus vector, SemlikiForrest virus, a poxvirus, a papilloma virus, a retrovirus or alentivirus. Additional vectors used to express IcsP and/or SigA includevirus-like particles (VLP) and bacteriophages.

Additionally, IcsP and SigA can be incorporated into a a recombinantexpression vector comprising a selection gene, a yeast sequence, and apolynucleotide encoding IcsP and/or SigA, wherein said polynucleotide isoperably linked to a yeast promoter and said vector is being used totransfect yeast cells which produce IcsP and/or SigA polypeptides of thepresent invention. The recombinant expression vectors of the inventioncan be designed for expression of the proteins of the invention in yeastcells. Methods of expressing proteins in yeast, such as Saccharomycescerevisiae, Pichia pastoris, Hansenula polymorpha, and Kluyveromyceslactis, are well-known in the art.

The present invention also relates to the use of reagents specific forthe IcsP and SigA genes and the IcsP2 and SigA2 polypeptides in thedesign of diagnostic tests.

In a further embodiment of the invention the antigens disclosed arecloned and expressed in non virulent or in an attenuated bacteria andthe later are used as vectors containing a DNA promoter element capableof initiating the synthesis of mRNA operably linked to an open readingframe containing one or both of the genes encoding Shigella IcsP andSigA. The resultant protein(s) is(are) exported and assembled on thebacterial surface and/or periplasm. Such non virulent or attenuatedbacteria can then be used as oral or mucosal vaccine. In anotherembodiment, the IcsP and the SigA antigens are being overexpressed innon virulent strains or mutant strains of Shigella that have beenequipped with a suitable promoter. Such bacteria expressing either IcsPor SigA antigens or both can then be used as live vaccines againstshigellosis. Alternatively, such overexpressing strains can beinactivated with formalin or by heating and the resulting bacteria canbe used as killed vaccines. Further embodiments of the invention arevectors used to transform Shigella species which results in theperiplasmic expression of heterologous antigens. This expression is notlikely to alter either Shigella's natural tissue tropism (colonicepithelium) following oral administration or significantly reduce straininvasiveness. Suitable Shigella species include live, attenuated vaccinestrains of S. sonnei, S. dysenteriae, Sflextieri, and S. boydii.Exemplified transformed Shigella strains include Shigella vaccinestrain, e.g. Shigella flexneri 2a (SC608(3098)), Shigella flexneri 2a(SC608(cfaAE)), Shigella flexneri 2a (SC608(pCFAI)) and Shigellaflexneri 2a (SC608(pCFAI/LTB)). These strains are characterized ashaving deletions in icsA, a gene that enables intracellular andintercellular spread of Shigella in host epithelial cells and in thegene iucA that plays a role in iron acquisition by the bacteria. Thesetransformed Shigella strains are suitable for use in immunogeniccomposition, in particular oral or mucosally administered vaccines.Other bacteria have been described and are well known in the art for useas vector systems:

-   -   Salmonella and E. coli bacterial surface proteins have been used        as carriers or vehicles of foreign epitopes for various        purposes, including the development of live vaccines (U.S. Pat.        No. 5,348,867, Inventors Georgiou, George, Francisco, Joseph A,        Earhart, Charles F.)    -   Lactobacillus harboring an expression cassette encoding a signal        sequence, wherein the biologically active polypeptide is linked        to a heterologous carboxy-terminal target region.        (WO/2005/012491, PCT/US2004/002460) Inventors: CHANG, Chia-Hwa,        LIU, Xiaowen et al.)    -   Bacterial surface protein expression: Smit, John; and, Nina        Agabian; “Cloning of the Major Protein of the Caulobacter        crescentus Periodic Surface Layer Detection and Characterization        of the Cloned Peptide by Protein Expression Assays” (1984) J.        Bacteriol. 160, 1137-1145. U.S. Pat. No. 5,500,353.    -   Compartmentalization of recombinant polypeptides in host cells.        (PCT/EP00/00686, U.S. Pat. No. 6,610,517, Inventor Werner        Lubitz).    -   Yeast cell surface display of proteins and uses thereof (U.S.        Pat. No. 6,423,538) Wittrup, K. Dane et al.).    -   Recombinant mycobacteria, particularly recombinant M. bovis BCG,        which express heterologous DNA encoding a product (protein or        polypeptide) of interest (U.S. Pat. No. 5,591,632) O'Donnell,        Michael A. et al.)    -   Use of gram-positive bacteria to express recombinant proteins        (U.S. Pat. No. 5,821,088) Darzins, Aldis et al. Gianni Pozzi et        al., “Delivery and Expression of a Heterologous Antigen on the        Surface of Streptococci”, Infection and Immunity (May 1992)        60:1902-1907    -   Method for expression and secretion in bacillus (U.S. Pat. No.        5,032,510)—S. Kovacevic et al.

The present invention also relates to the use of reagents specific forthe IcsP and SigA genes and the IcsP2 and SigA2 polypeptides in thedesign of diagnostic tests.

The technique of gene amplification referred to as polymerase chainreaction (PCR) is well known in the art and has been used for thediagnosis of Shigella infections (41). The present invention disclosesspecific primers capable of binding to the IcsP2 and the SigA2 genesequences. Such primers can be used to amplify either icsP or sigA genesfrom bacteria present in a clinical sample (e.g. stool) and theamplified fragment can be detected by visual, photometric, isotopic orfluorometric methods. Thus, the present invention claims the use ofoligonucleotide primers specific for said IcsP2 and SigA2 gene sequencesin the diagnosis of dysentery caused by Shigella spp and EIEC.

The present invention also relates to the production of antisera againstSigA2 and IcsP2. Such antisera were capable of reacting withcorresponding polypeptides expressed by different species of Shigella.Further, such antisera were capable of inhibiting in vitro infection ofHeLa cells by Shigella bacteria and of protecting animals againstinflammation caused by Shigella, in a keratoconjunctivitis model.

The present invention also relates to conjugates of IscP2 and an Opolysaccharide antigen of Shigella and conjugates of the SigA2 and of Opolysaccharide antigen of Shigella. The O-specific polysaccharides onthe surface of pathogenic bacteria are thought to be both protectiveantigens and essential virulence factors. The inability of mostpolysaccharides to elicit protective levels of anti-polysaccharideantibodies in infants and adults with weakened immune systems could beovercome by their covalent attachment to proteins that conferred T-celldependent properties. This principle led to the construction of vaccinesagainst Haemophilus influenzae b (Hib), pneumococcal pneumonia, andNeisseria meningitidis. Extension of the conjugate technology to theO-specific polysaccharides of Gram-negative bacteria provided a newgeneration of glycoconjugate vaccines. Originally, Avery and Goebel inJ. Exp. Med. 50:531 (1929) and Goebel in J. Exp. Med. 50:469-520 (1929)showed that the immunogenicity of pneumococcus type 3 polysaccharidecould be increased by binding it chemically to a carrier protein. Thisprinciple has been applied successfully to increase the immunogenicityof polysaccharides of other pathogens. Methods to couple covalentlypolysaccharides to protein carriers are known in the art and include thefollowing:

Gu, X., et al., “Synthesis, Characterization, and Immunologic Propertiesof Detoxified Lipooligosaccharide from Nontypeable Haemophilus influenzaConjugated to Proteins”, Infection and Immunity, 64(10), (1996) pp.4047-4053.

Gupta, R., et al., “Comparative Immunogenicity of Conjugates Composed ofEscherichia coli O111 O-Specific Polysaccharide, Prepared by Treatmentwith Acetic Acid or Hydrazine, Bound to Tetanus Toxoid by Two SyntheticSchemes”, Infection and Immunity, 63(8), (1995), pp. 2805-2810.

Gupta, R., et al., “Synthesis, Characterization, and Some ImmunologicalProperties of Conjugates Composed of the Detoxified Lipopolysaccharideof Vibrio cholerae O1 Serotype Inaba Bound to Cholera Toxin”, Infectionand Immunity, 60(8), (1992), pp. 3201-3208.

-   Konadu, E., et al., “Investigational Vaccine for Escherichia coli    0157: Phase 1 Study of O157 O-Specific Polysaccharide-Pseudomonas    aeruginosa Recombinant Exprotein A Conjugates in Adults”, Journal of    Infectious Diseases, 177, (1998), pp. 383-387.-   Konadu, E., et al., “Phase 1 and Phase 2 Studies of Salmonella    enterica Serovar Paratyphi A O-Specific Polysaccharide-Tetanus    Toxoid Conjugates in Adults, Teenagers, and 2- to 4-Year Old    Children in Vietnam”, Infection and Immunity, 68(3), (2000), pp.    1529-1534.-   Konadu, E., et al., “Preparation, Characterization, and    Immunological Properties in Mice of Escherichia coli O157 O-Specific    Polysaccharide-Protein Conjugate Vaccines”, Infection and Immunity,    62(11), (1994), pp. 5048-5054.-   Robbins, J., et al., “Polysaccharide-Protein Conjugates: A New    Generation of Vaccines”, The Journal of Infectious Diseases, 161,    (1990), pp. 821-832.-   Taylor, D., et al., “Synthesis, Characterization, and Clinical    Evaluation of Conjugate Vaccines Composed of the O-Specific    Polysaccharides of Shigella dysenteriae Type 1, Shigella flexneri    Type 2a, and Shigella sonnei (Plesiomonas shigelloides) Bound to    Bacterial Toxoids”, Infection and Immunity, 61, (1993), pp.    3678-3687.

WO/1999/003871; U.S. Pat. No. 4,771,127; U.S. Pat. No. 5,866,132.

In another embodiment, the invention relates to conjugates whereinShigella O polysaccharide is coupled through a spacer to either IcsP orSigA to enhance antigenicity and immunogenicity of the polysaccharideand the conjugate is used to induce an immune response to both theprotein and the polysaccharide.

In yet another embodiment, the Shigella IcsP and SigA proteins can bechemically conjugated or are the products of genetic fusion with otherproteins for use as immunogens in, for example, vaccines. Such proteinsinclude tetanus toxoid, diptheria toxoid, cholera toxin B subunit, E.coli enterotoxin B subunit, and flagellin. These proteins are well knownin the art and have been extensively used for these purposes in theindustry and/or research as set forth in the following publications:

-   S J McKenzie and J F Halsey Cholera toxin B subunit as a carrier    protein to stimulate a mucosal immune response. The Journal of    Immunology, Vol 133, Issue 4 1818-1824,-   S. Shah, R. Raghupathy, Om. Singh, G. P. Talwar- and A. Sodhi. Prior    immunity to a carrier enhances antibody responses to hCG in    recipients of an hCG-carrier conjugate vaccine. Vaccine, Volume 17,    Issues 23-24, 1999, Pages 3116-3123-   LE MOIGNE Vincent; ROBREAU Georges; MAHANA Wahib; Flagellin as a    good carrier and potent adjuvant for Th1 response: Study of mice    immune response to the p27 (Rv2108) Mycobacterium tuberculosis    antigen. Molecular immunology 2008, vol. 45, no 9, pp. 2499-2507-   Camilo Cuadros, Francisco J. Lopez-Hernandez, Ana Lucia Dominguez,    Michael McClelland, and Joseph Lustgarten. Flagellin Fusion Proteins    as Adjuvants or Vaccines Induce Specific Immune Responses. Infection    and Immunity, May 2004, p. 2810-2816, Vol. 72, No. 5.

In yet another embodiment, the invention relates to conjugates whereinthe polysaccharide of another enteropathogenic bacteria such asSalmonella Typhi Vi polysaccharide or Salmonella Paratyphi is covalentlycoupled through a spacer to either IcsP or SigA, and the conjugate isused to induce an immune response to both the protein and thepolysaccharide and thus to vaccinate against both shigellosis andtyphoid (or paratyphoid) disease.

The present invention also provides methods to produce anti-ShigellaIcsP2 and SigA2 antibodies in animals and recombinant polypeptides foruse in diagnostic methods for detecting Shigella in patients known orsuspected of having shigellosis. Such antibodies present in the sera ofimmunized mice and guinea pigs can also be produced in other animalspecies, such as horse, goat, rabbit, monkey, cattle, donkey, hamster.Molecular biology and antibody technology, such as that involving theuse of hybridomas, has made available to researchers and clinicianssources of highly specific and potent monoclonal antibodies useful ingeneral diagnostic and clinical procedures. Such monoclonal antibodiescan be obtained by standard fusion of immune cells from an animalimmunized with either IcsP2 or SigA2 with appropriate myeloma cells.More specifically, nucleic acid, protein or peptide molecules of theinvention may be utilized to develop monoclonal or polyclonal antibodiesthat bind Shigella IcsP2 or SigA2. For preparation of the ShigellaIcsP2- or SigA2-binding antibodies of the present invention, anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture may be used. For example, the hybridomatechnique originally developed by Kohler and Milstein (256 Nature495-497 (1975)) may be used. See also U.S. Pat. No. 4,376,110; Ausubelet al., Antibodies: a Laboratory Manual, (Harlow & Lane eds., ColdSpring Harbor Lab. 1988); Current Protocols in Immunology, (Colligan etal., eds., Greene Pub. Assoc. & Wiley Interseience N.Y., 1992-1996).

Another advantageous route for creating high affinity and/or highavidity human antibodies involves antigen priming of native humanlymphocytes in vitro, transferral of the resultant in vitro antigenprimed lymphocytes to an immunocompromised donor, e.g., a SCID mouse,boosting the immunocompromised donor with antigen, isolating humanantibody secreting B-cells (IgG secreting) from the donor, andEBV-transforming the isolated human antibody secreting cells, asdescribed in U.S. Pat. No. 6,537,809.

The antibodies of the present invention include chimeric antibodiescomprising part human and part mouse antibodies, in which the constantregion from human antibodies are cloned to a variable regions of lightand heavy chains from mouse. In some instances, 70% of the humansequences are retained. Humanized antibodies are chimeric antibodies inwhich perhaps 90% of the human antibody framework is retained, andcombined only with the murine the complementary determining regions.Fully humanized antibodies are also contemplated in the presentinvention.

Recombinant murine or chimeric murine-human or human-human antibodiesthat bind an epitope included in the amino acid sequences of ShigellaIcsP2 or SigA2 can be provided using known techniques. See, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. WileyInterscience, N.Y., 1987, 1992, 1993); Sambrook et al. MolecularCloning: A Laboratory Manual (Cold Spring Harbor Lab. Press 1989);EP0239400.

Anti-Shigella IcsP2 and SigA2 antibodies and/or peptides of the presentinvention are useful for immunoassays which detect or quantitateShigella IcsP2 and SigA2, or anti-Shigella IcsP2 and SigA2 antibodies,in a sample. An immunoassay for Shigella IcsP2 and SigA2 typicallycomprises incubating a clinical or biological sample in the presence ofa detectably labeled high affinity (or high avidity) anti-Shigella IcsP2or SigA2 antibody or polypeptide of the present invention capable ofselectively binding to IcsP2-specific antibodies or SigA2-specificantibodies, and detecting the labeled peptide or antibody which is boundin a sample. Various clinical assay procedures are well known in theart. See, e.g., Immunoassays for the 80's (Voller et al., eds.,University Park, 1981). Such samples include tissue blood, serum, andfecal samples, or liquids collected from the colorectal track followingenema or oral laxative solution and subjected to ELISA analysis asdescribed below.

Thus, an anti-Shigella IcsP2 or SigA2 antibodies or Shigella IcsP2 andSigA2 polypeptides can be fixed to nitrocellulose, or another solidsupport which is capable of immobilizing soluble proteins. The supportcan then be washed with suitable buffers followed by treatment with thedetectably labeled Shigella IcsP2 and SigA2-specific peptide orantibody. The solid phase support can then be washed with the buffer asecond time to remove unbound peptide or antibody. The amount of boundlabel on the solid support can then be detected by known method steps.

“Solid phase support” or “carrier” refers to any support capable ofbinding peptide, antigen, or antibody. Well-known supports or carriers,include glass, polystyrene, polypropylene, polyethylene, polyvinylfluoride (PVDF), dextran, nylon, amylases, natural and modifiedcelluloses, polyacrylamides, agaroses, and magnetite. The nature of thecarrier can be either soluble to some extent or insoluble for thepurposes of the present invention. The support material can havevirtually any possible structural configuration so long as the coupledmolecule is capable of binding to Shigella IcsP2 and SigA2 or ananti-Shigella IcsP2 or anti-Shigella SigA2 antibody. Thus, the supportconfiguration can be spherical, as in a bead, or cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface can be flat, such as a sheet, culture dish,test strip, etc. For example, supports may include polystyrene beads.Those skilled in the art will know many other suitable carriers forbinding antibody, peptide or antigen, or can ascertain the same byroutine experimentation.

Well known method steps can determine binding activity of a given lot ofanti-Shigella IcsP2 and SigA2 peptide and/or antibody. Those skilled inthe art can determine operative and optimal assay conditions by routineexperimentation.

Detectably labeling a Shigella IcsP2- or SigA2-specific peptide and/orantibody can be accomplished by linking to an enzyme for use in anenzyme immunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA).The linked enzyme reacts with the exposed substrate to generate achemical moiety which can be detected, for example, byspectrophotometric, fluorometric or by visual means. Enzymes which canbe used to detectably label the Shigella IcsP2- and SigA2-specificantibodies of the present invention include, but are not limited to,horseradish peroxidase, alkaline phosphatase, glucose oxidase,beta-galactosidase, glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

By radioactively labeling the Shigella IcsP2- and SigA2-specificantibodies, it is possible to detect Shigella IcsP2 and SigA2 throughthe use of a radioimmunoassay (RIA). See Work et al., LABORATORYTECHNIQUES & BIOCHEMISTRY IN MOLECULAR BIOLOGY (North Holland PublishingCo., N.Y. (1978). The radioactive isotope can be detected by such meansas the use of a gamma counter or a scintillation counter or byautoradiography. Isotopes which are particularly useful for the purposeof the present invention are: 3H, 125I, 131I, 35S, 14C, and 125I.

It is also possible to label the Shigella IcsP2- and SigA2-specificantibodies with a fluorescent compound. When the fluorescent labeledantibody is exposed to light of the proper wave length, its presence canthen be detected due to fluorescence. Among the most commonly usedfluorescent labelling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine.

The Shigella IcsP2 and SigA2-specific antibodies can also be detectablylabeled using fluorescence-emitting metals such as 125Eu, or others ofthe lanthanide series. These metals can be attached to the ShigellaIcsP2- and SigA2-specific antibodies using such metal chelating groupsas diethylenetriaminepentaacetic acid (DTPA) orethylenediamine-tetraacetic acid (EDTA).

The Shigella IcsP2- and SigA2-specific antibodies also can be detectablylabeled by coupling to a chemiluminescent compound. The presence of thechemiluminescently labeled antibody is then determined by detecting thepresence of luminescence that arises during the course of a chemicalreaction. Examples of useful chemiluminescent labeling compounds areluminol, isoluminol, theromatic acridinium ester, imidazole, acridiniumsalt and oxalate ester.

Likewise, a bioluminescent compound can be used to label the ShigellaIcsP2- and SigA2-specific antibody, portion, fragment, polypeptide, orderivative of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

Detection of the Shigella IcsP2- and SigA2-specific antibodies, portion,fragment, polypeptide, or derivative can be accomplished by ascintillation counter, for example, if the detectable label is aradioactive gamma emitter, or by a fluorometer, for example, if thelabel is a fluorescent material. In the case of an enzyme label, thedetection can be accomplished by colorometric methods which employ asubstrate for the enzyme. Detection can also be accomplished by visualcomparison of the extent of enzymatic reaction of a substrate incomparison with similarly prepared standards.

For the purposes of the present invention, the Shigella IcsP2 and SigA2which is detected by the above assays can be present in a biologicalsample. Any sample containing Shigella IcsP2 or SigA2 can be used. Forexample, the sample is a biological fluid such as, for example, blood,serum, urine, feces, a tissue extract or homogenate, and the like.However, the invention is not limited to assays using only thesesamples, it being possible for one of ordinary skill in the art todetermine suitable conditions which allow the use of other samples.

The antibody, fragment or derivative of the present invention can beadapted for utilization in an immunometric assay, also known as a“two-site” or “sandwich” assay. In a typical immunometric assay, aquantity of unlabeled antibody (or fragment of antibody) is bound to asolid support that is insoluble in the fluid being tested and a quantityof detectably labeled soluble antibody is added to permit detectionand/or quantitation of the ternary complex formed between solid-phaseantibody, antigen, and labeled antibody.

Typical, immunometric assays include “forward” assays in which theantibody bound to the solid phase is first contacted with the samplebeing tested to extract the Shigella IcsP2- or SigA2-containing proteinsfrom the sample by formation of a binary solid phase antibody-ShigellaIcsP2 or SigA2 complex. After a suitable incubation period, the solidsupport is washed to remove the residue of the fluid sample, includingunreacted Shigella IcsP2 or SigA2, if any, and then contacted with thesolution containing a known quantity of labeled antibody (whichfunctions as a “reporter molecule”). After a second incubation period topermit the labeled antibody to complex with the Shigella IcsP2 or SigA2bound to the solid support through the unlabeled antibody, the solidsupport is washed a second time to remove the unreacted labeledantibody. This type of forward sandwich assay can be used to determinewhether Shigella IcsP2 and/or SigA2 is present or can be madequantitative by comparing the measure of labeled antibody with thatobtained for a standard sample containing known quantities of ShigellaIcsP2 and SigA2. Such “two-site” or “sandwich” assays are described byWide, Radioimmune Assay Methods, 199-206 (Kirkham, ed., Livingstone,Edinburgh, 1970).

Other type of “sandwich” assays, which can also be useful with ShigellaIcsP2 and SigA2, are the so-called “simultaneous” and “reverse” assays.A simultaneous assay involves a single incubation step wherein theantibody bound to the solid support and labeled antibody are both addedto the sample being tested at the same time. After the incubation iscompleted, the solid support is washed to remove the residue of fluidsample and uncomplexed labeled antibody. The presence of labeledantibody associated with the solid support is then determined as itwould be in a conventional sandwich assay.

In the “reverse” assay, stepwise addition first of a solution of labeledantibody to the fluid sample followed by the addition of unlabeledantibody bound to a solid support after a suitable incubation period, isutilized. After a second incubation, the solid phase is washed inconventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support is then determinedas in the “simultaneous” and “forward” assays. In one embodiment, acombination of antibodies of the present invention specific for separateepitopes can be used to construct a sensitive three-siteimmunoradiometric assay.

In accordance with the present invention there may be numerous tools andtechniques within the skill of the art, such as those commonly used inmolecular immunology, cellular immunology, pharmacology, andmicrobiology. Such tools and techniques are described in detail in e.g.,Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed.Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Ausubelet al. eds. (2005) Current Protocols in Molecular Biology. John Wileyand Sons, Inc.: Hoboken, N.J.; Bonifacino et al. eds. (2005) CurrentProtocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.;Coligan et al. eds. (2005) Current Protocols in Immunology, John Wileyand Sons, Inc.: Hoboken, N.J.; Coico et al. eds. (2005) CurrentProtocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.;Coligan et al. eds. (2005) Current Protocols in Protein Science, JohnWiley and Sons, Inc.: Hoboken, N.J.; and Enna et al. eds. (2005) CurrentProtocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, N.J.

The abbreviations in the specification correspond to units of measure,techniques, properties or compounds as follows: “min” means minutes, “h”means hour(s), “μL” means microliter(s), “mL” means milliliter(s), “mM”means millimolar, “M” means molar, “mmole” means millimole(s), “kb”means kilobase, “bp” means base pair(s), and “IU” means InternationalUnits.

“Polymerase chain reaction” is abbreviated PCR; “Reverse transcriptasepolymerase chain reaction” is abbreviated RT-PCR; “Untranslated region”is abbreviated UTR; “Sodium dodecyl sulfate” is abbreviated SDS; and“High Pressure Liquid Chromatography” is abbreviated HPLC.

“Amplification” of DNA as used herein denotes the use of polymerasechain reaction (PCR) to increase the concentration of a particular DNAsequence within a mixture of DNA sequences. For a description of PCR seeSaiki et al., Science 1988, 239:487.

A “polynucleotide” or “nucleotide sequence” is a series of nucleotidebases (also called “nucleotides”) in a nucleic acid, such as DNA andRNA, and means any chain of two or more nucleotides. A nucleotidesequence typically carries genetic information, including theinformation used by cellular machinery to make proteins and enzymes.These terms include double or single stranded genomic and cDNA, RNA, anysynthetic and genetically manipulated polynucleotide, and both sense andanti-sense polynucleotide (although only sense stands are beingrepresented herein). This includes single- and double-strandedmolecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as“protein nucleic acids” (PNA) formed by conjugating bases to an aminoacid backbone. This also includes nucleic acids containing modifiedbases, for example thio-uracil, thio-guanine and fluoro-uracil.

The nucleic acids herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, andinternucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters,phosphoroamidates, carbamates, etc.) and with charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.). Polynucleotides maycontain one or more additional covalently linked moieties, such as, forexample, proteins (e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, iron, oxidative metals,etc.), and alkylators. The polynucleotides may be derivatized byformation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the polynucleotides herein mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin, and the like.

The term “nucleic acid hybridization” refers to anti-parallel hydrogenbonding between two single-stranded nucleic acids, in which A pairs withT (or U if an RNA nucleic acid) and C pairs with G. Nucleic acidmolecules are “hybridizable” to each other when at least one strand ofone nucleic acid molecule can form hydrogen bonds with the complementarybases of another nucleic acid molecule under defined stringencyconditions. Stringency of hybridization is determined, e.g., by (i) thetemperature at which hybridization and/or washing is performed, and (ii)the ionic strength and (iii) concentration of denaturants such asformamide of the hybridization and washing solutions, as well as otherparameters. Hybridization requires that the two strands containsubstantially complementary sequences. Depending on the stringency ofhybridization, however, some degree of mismatches may be tolerated.Under “low stringency” conditions, a greater percentage of mismatchesare tolerable (i.e., will not prevent formation of an anti-parallelhybrid). See Molecular Biology of the Cell, Alberts et al., 3rd ed., NewYork and London: Garland Publ., 1994, Ch. 7.

Typically, hybridization of two strands at high stringency requires thatthe sequences exhibit a high degree of complementarity over an extendedportion of their length. Examples of high stringency conditions include:hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at65° C., followed by washing in 0.1×SSC/0.1% SDS at 68° C. (where 1×SSCis 0.15M NaCl, 0.15M Na citrate) or for oligonucleotide moleculeswashing in 6×SSC/0.5% sodium pyrophosphate at about 37° C. (for 14nucleotide-long oligos), at about 48° C. (for about 17 nucleotide-longoligos), at about 55° C. (for 20 nucleotide-long oligos), and at about60° C. (for 23 nucleotide-long oligos)). Accordingly, the term “highstringency hybridization” refers to a combination of solvent andtemperature where two strands will pair to form a “hybrid” helix only iftheir nucleotide sequences are almost perfectly complementary (seeMolecular Biology of the Cell, Alberts et al., 3rd ed., New York andLondon: Garland Publ., 1994, Ch. 7).

Conditions of intermediate or moderate stringency (such as, for example,an aqueous solution of 2×SSC at 65° C.; alternatively, for example,hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at65° C., and washing in 0.2×SSC/0.1% SDS at 42° C.) and low stringency(such as, for example, an aqueous solution of 2×SSC at 55° C.), requirecorrespondingly less overall complementarity for hybridization to occurbetween two sequences. Specific temperature and salt conditions for anygiven stringency hybridization reaction depend on the concentration ofthe target DNA and length and base composition of the probe, and arenormally determined empirically in preliminary experiments, which areroutine (see Southern, J. Mol. Biol. 1975; 98: 503; Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 2, ch. 9.50,CSH Laboratory Press, 1989; Ausubel et al. (eds.), 1989, CurrentProtocols in Molecular Biology, Vol. I, Green Publishing Associates,Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).

As used herein, the term “standard hybridization conditions” refers tohybridization conditions that allow hybridization of sequences having atleast 75% sequence identity. According to a specific embodiment,hybridization conditions of higher stringency may be used to allowhybridization of only sequences having at least 80% sequence identity,at least 90% sequence identity, at least 95% sequence identity, or atleast 99% sequence identity.

Nucleic acid molecules that “hybridize” to any desired nucleic acids ofthe present invention may be of any length. In one embodiment, suchnucleic acid molecules are at least 10, at least 15, at least 20, atleast 30, at least 40, at least 50, and at least 70 nucleotides inlength. In another embodiment, nucleic acid molecules that hybridize areof about the same length as the particular desired nucleic acid.

As used herein, the term “isolated” means that the referenced materialis removed from the environment in which it is normally found. Thus, anisolated biological material can be free of cellular components, i.e.,components of the cells in which the material is found or produced.Isolated nucleic acid molecules include, for example, a PCR product, anisolated mRNA, a cDNA, or a restriction fragment. Isolated nucleic acidmolecules also include, for example, sequences inserted into plasmids,cosmids, artificial chromosomes, and the like. An isolated nucleic acidmolecule is preferably excised from the genome in which it may be found,and more preferably is no longer joined to non-regulatory sequences,non-coding sequences, or to other genes located upstream or downstreamof the nucleic acid molecule when found within the genome. An isolatedprotein may be associated with other proteins or nucleic acids, or both,with which it associates in the cell, or with cellular membranes if itis a membrane-associated protein.

A “host cell” includes an individual cell or cell culture which can beor has been a recipient for vector(s) or for incorporation ofpolynucleotide molecules. In the present invention, a host cell can be abacteria, a mammalian cell, an insect cell or a yeast cell.

“Treating” or “treatment” of a state, disorder or condition includes:

(1) preventing or delaying the appearance of clinical or sub-clinicalsymptoms of the state, disorder or condition developing in a mammal thatmay be afflicted with or predisposed to the state, disorder or conditionbut does not yet experience or display clinical or subclinical symptomsof the state, disorder or condition; or(2) inhibiting the state, disorder or condition, i.e., arresting,reducing or delaying the development of the disease or a relapse thereof(in case of maintenance treatment) or at least one clinical orsub-clinical symptom thereof; or(3) relieving the disease, i.e., causing regression of the state,disorder or condition or at least one of its clinical or sub-clinicalsymptoms.

The benefit to a subject to be treated is either statisticallysignificant or at least perceptible to the patient or to the physician.

An “immune response” refers to the development in the host of a cellularand/or antibody-mediated immune response to a composition or vaccine ofinterest. Such a response usually consists of the subject producingantibodies, B cells, helper T cells, and/or cytotoxic T cells directedspecifically to an antigen or antigens included in the composition orvaccine of interest. The immune response also may include regulatoryT-cells, whose activity is beyond the organism of interest, and maysuppress other immune or allergic responses.

A “therapeutically effective amount” means the amount of a compound,adjuvant, or vaccine composition that, when administered to a mammal fortreating a state, disorder or condition, is sufficient to effect suchtreatment. The “therapeutically effective amount” will vary depending onthe compound, bacteria or analogue administered as well as the diseaseand its severity and the age, weight, physical condition andresponsiveness of the mammal to be treated.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

While it is possible to use a composition provided by the presentinvention for therapy as is, it may be preferable to administer it in apharmaceutical formulation, e.g., in admixture with a suitablepharmaceutical excipient, diluent or carrier selected with regard to theintended route of administration and standard pharmaceutical practice.Accordingly, in one aspect, the present invention provides apharmaceutical composition or formulation comprising at least one activecomposition, or a pharmaceutically acceptable derivative thereof, inassociation with a pharmaceutically acceptable excipient, diluent and/orcarrier. The excipient, diluent and/or carrier must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The compositions of the invention can be formulated for administrationin any convenient way for use in human or veterinary medicine. Theinvention therefore includes within its scope pharmaceuticalcompositions comprising a product of the present invention that isadapted for use in human or veterinary medicine.

In a preferred embodiment, the pharmaceutical composition isconveniently administered as a liquid oral formulation. Although thereare no physical limitations to delivery of the formulation, oraldelivery is preferred because of its ease and convenience, and becauseoral formulations readily accommodate additional mixtures, such as milk,yoghurt, and infant formula. Other oral dosage forms are well known inthe art and include tablets, caplets, gelcaps, capsules, and medicalfoods. Tablets, for example, can be made by well-known compressiontechniques using wet, dry, or fluidized bed granulation methods.

Such oral formulations may be presented for use in a conventional mannerwith the aid of one or more suitable excipients, diluents, and carriers.Pharmaceutically acceptable excipients assist or make possible theformation of a dosage form for a bioactive material and includediluents, binding agents, lubricants, glidants, disintegrants, coloringagents, and other ingredients. Preservatives, stabilizers, dyes and evenflavoring agents may be provided in the pharmaceutical composition.Examples of preservatives include sodium benzoate, ascorbic acid andesters of p-hydroxybenzoic acid. Antioxidants and suspending agents maybe also used. An excipient is pharmaceutically acceptable if, inaddition to performing its desired function, it is non-toxic, welltolerated upon ingestion, and does not interfere with absorption ofbioactive materials.

Acceptable excipients, diluents, and carriers for therapeutic use arewell known in the pharmaceutical art, and are described, for example, inRemington: The Science and Practice of Pharmacy. Lippincott Williams &Wilkins (A. R. Gennaro edit. 2005). The choice of pharmaceuticalexcipient, diluent, and carrier can be selected with regard to theintended route of administration and standard pharmaceutical practice.

As used herein, the phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that are generally regarded asphysiologically tolerable.

“Patient” or “subject” refers to mammals and includes human andveterinary subjects.

The dosage of an adjuvant formulation or vaccine composition containingthe adjuvant will vary widely, depending upon the nature of the disease,the patient's medical history, the frequency of administration, themanner of administration, the clearance of the agent from the host, andthe like. The initial dose may be larger, followed by smallermaintenance doses. The dose may be administered as infrequently asmonthly or annually to maintain an effective immunological memory.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water or aqueoussolution saline solutions and aqueous dextrose and glycerol solutionsare preferably employed as carriers, particularly for injectablesolutions. Alternatively, the carrier can be a solid dosage formcarrier, including but not limited to one or more of a binder (forcompressed pills), a glidant, an encapsulating agent, a flavorant, and acolorant. Suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E.W. Martin.

The invention also encompasses pharmaceutical compositions and vaccines.The pharmaceutical compositions and vaccine compositions of theinvention comprise at least one of the novel Shigella antigens, and oneor more adjuvants along with a pharmaceutically acceptable carrier orexcipient. Methods of formulating pharmaceutical compositions andvaccines are well-known to those of ordinary skill in the art, asdescribed in Remington's, supra.

Formulations. The compositions of the present invention may comprisepharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength; additives such as detergents and solubilizingagents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimersol, benzylalcohol) and bulking substances (e.g., lactose, mannitol);incorporation' of the material into particulate preparations ofpolymeric compounds such as polylactic acid, polyglycolic acid, etc. orinto liposomes. Hylauronic acid may also be used. See, e.g., Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa. 18042) pages 1435-1712 which are herein incorporated by reference.

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets, pellets, powders, orgranules. Also, liposomal or proteinoid encapsulation may be used toformulate the present compositions (as, for example, proteinoidmicrospheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation may be used and the liposomes may be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given by Marshall, K.In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter10, 1979, herein incorporated by reference. In general, the formulationwill include the therapeutic agent and inert ingredients which allow forprotection against the stomach environment, and release of thebiologically active material in the intestine.

Also contemplated for use herein are liquid dosage forms for oraladministration, including pharmaceutically acceptable emulsions,solutions, suspensions, and syrups, which may contain other componentsincluding inert diluents; adjuvants, wetting agents, emulsifying andsuspending agents; and sweetening, flavoring, coloring, and perfumingagents.

For oral formulations, the location of release may be the stomach, thesmall intestine (the duodenum, the jejunem, or the ileum), or the largeintestine. One skilled in the art has available formulations which willnot dissolve in the stomach, yet will release the material in theduodenum or elsewhere in the intestine, e.g., by the use of an entericcoating. Examples of the more common inert ingredients that are used asenteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic (i.e. powder), for liquid forms a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used. The formulation of the materialfor capsule administration could also be as a powder, lightly compressedplugs, or even as tablets. These therapeutics could be prepared bycompression.

One may dilute or increase the volume of the therapeutic agent with aninert material. These diluents could include carbohydrates, especiallymannitol, -lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeuticagent into a solid dosage form. Materials used as disintegrates includebut are not limited to starch, including the commercial disintegrantbased on starch, Explotab, Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. The disintegrants may also be insoluble cationicexchange resins. Powdered gums may be used as disintegrants and asbinders and can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrantsBinders may be used to hold the therapeutic agent togetherto form a hard tablet and include materials from natural products suchas acacia, tragacanth, starch and gelatin. Others include methylcellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC).Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC)could both be used in alcoholic solutions to granulate the peptide (orderivative).

An antifrictional agent may be included in the formulation to preventsticking during the formulation process. Lubricants may be used as alayer between the peptide (or derivative) and the die wall, and thesecan include but are not limited to; stearic acid including its magnesiumand calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin,vegetable oils and waxes. Soluble lubricants may also be used such assodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol ofvarious molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties drug during formulationand to aid rearrangement during compression might be added. The glidantsmay include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic agent into the aqueous environmenta surfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Controlled release oral formulations may used in practicing the presentinvention. The therapeutic agent could be incorporated into an inertmatrix which permits release by either diffusion or leaching mechanisms,e.g., gums. Slowly degenerating matrices may also be incorporated intothe formulation. Some enteric coatings also have a delayed releaseeffect. Another form of a controlled release is by a method based on theOros therapeutic system (Alza Corp.), i.e. the therapeutic agent isenclosed in a semipermeable membrane which allows water to enter andpush agent out through a single small opening due to osmotic effects.

Other coatings may be used for the formulation. These include a varietyof sugars which could be applied in a coating pan. The therapeutic agentcould also be given in a film coated tablet and the materials used inthis instance are divided into 2 groups. The first are the nonentericmaterials and include methyl cellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid. A mix ofmaterials might be used to provide the optimum film coating. Filmcoating may be carried out in a pan coater or in a fluidized bed or bycompression coating.

In one embodiment, the Shigella polypeptide antigens disclosed in thepresent invention are administered with a pharmaceutically acceptablediluent. Such formulations can be administered by an injection(subcutaneous, intradermal, intramuscular) or applied topically onto theskin using an adhesive patch. Alternatively, the vaccine is administeredby a mucosal route (oral, buccal, sublingual, nasal drops, aerosol,rectal) using a pharmaceutically acceptable vehicle. The antigens canalso be mixed with an adjuvant to enhance the ensuing immune responses.Example of such adjuvants are without being limited to, aluminium salts,ISCOMs, saponin-based adjuvants, oil-in-water and water-in-oilemulsions, toll-like receptor ligands such as muramyl dipeptide, E. coliLPS, oligonucleotides comprised of unmethylated DNA, poly I:C,lipoteichoic acid, peptidoglycan. Enterotoxins and their adjuvant activederivatives such as cholera toxin, heat-labile E. coli enterotoxin,pertussis toxin, shiga toxin and analogs

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants, preserving, wetting,emulsifying, and dispersing agents. The pharmaceutical compositions maybe sterilized by, for example, filtration through a bacteria retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions. They canalso be manufactured using sterile water, or some other sterileinjectable medium, immediately before use.

Vaccines. In the case of vaccines, it is often observed that a primarychallenge with an antigen alone, in the absence of an adjuvant, willfail to elicit a humoral or cellular immune response. Therefore thevaccines of the invention may contain adjuvants including, but notlimited to, cholera toxin, fragments and mutants or derivatives withadjuvant properties, Escherichia coli heat-labile enterotoxin, fragmentsand mutants or derivatives with adjuvant properties, oil-in-water andwater-in-oil emulsions, toll-like receptor ligands such as muramyldipeptide, E. coli LPS, oligonucleotides comprised of unmethylated DNA,poly I:C, lipoteichoic acid, peptidoglycan. Enterotoxins and theiradjuvant active derivatives such as cholera toxin, heat-labile E. colienterotoxin, pertussis toxin, shiga toxin and analogs. Other adjuvantscan be used such as complete Freund's adjuvant, incomplete Freund'sadjuvant, saponin, mineral gels such as aluminum hydroxide, surfaceactive substances such as lysolecithin, pluronic polyols, polyanions,peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, andpotentially useful human adjuvants such asN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine,BCG (bacille Calmette-Guerin) and Corynebacterium parvum. An adjuvantcan serve as a tissue depot that slowly releases the antigen and also asa lymphoid system activator that non-specifically enhances the immuneresponse (Hood et al., Immunology, Second Ed., 1984, Benjamin/Cummings:Menlo Park, Calif., p. 384). Where the vaccine is intended for use inhuman subjects, the adjuvant should be pharmaceutically acceptable.

Administration. Such pharmaceutical compositions or vaccines may be foradministration by oral (solid or liquid), parenteral (intramuscular,intraperitoneal, intravenous (IV) or subcutaneous injection),transdermal (either passively or using ionophoresis or electroporation),transmucosal (nasal, vaginal, rectal, or sublingual), or inhalationroutes of administration, or using bioerodible inserts and can beformulated in dosage forms appropriate for each route of administration.

In one preferred embodiment, the compositions or vaccines areadministered by pulmonary delivery. The composition or vaccine isdelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream [see, e.g., Adjei, et al.Pharmaceutical Research 1990; 7:565-569; Adjei, et al. Int. J.Pharmaceutics 1990; 63:135-144 (leuprolide acetate); Braquet, et al. J.Cardiovascular Pharmacology 1989; 13(sup5):143-146 (endothelin-1);Hubbard, et al. (1989) Annals of Internal Medicine, Vol. III, pp.206-212 (α1-antitrypsin); Smith, et al. J. Clin. Invest. 1989;84:1145-1146 (α-1-proteinase); Oswein, et al. “Aerosolization ofProteins”, 1990; Proceedings of Symposium on Respiratory Drug DeliveryII Keystone, Colo. (recombinant human growth hormone); Debs, et al. J.Immunol. 1988; 140:3482-3488 (interferon-γ and tumor necrosis factor α);and U.S. Pat. No. 5,284,656 to Platz, et al. (granulocyte colonystimulating factor). A method and composition for pulmonary delivery ofdrugs for systemic effect is described in U.S. Pat. No. 5,451,569 toWong, et al. See also U.S. Pat. No. 6,651,655 to Licalsi et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer(Marquest Medical Products, Englewood, Colo.); the Ventolin metered doseinhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhalerpowder inhaler (Fisons Corp., Bedford, Mass.). All such devices requirethe use of formulations suitable for the dispensing of the therapeuticagent. Typically, each formulation is specific to the type of deviceemployed and may involve the use of an appropriate propellant material,in addition to the usual diluents, adjuvants, surfactants and/orcarriers useful in therapy. Also, the use of liposomes, microcapsules ormicrospheres, inclusion complexes, or other types of carriers iscontemplated.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the therapeutic agentsuspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the therapeutic agent, and may alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, or mannitolin amounts which facilitate dispersal of the powder from the device,e.g., 50 to 90% by weight of the formulation. The therapeutic agentshould most advantageously be prepared in particulate form with anaverage particle size of less than 10 mm (or microns), most preferably0.5 to 5 mm, for most effective delivery to the distal lung.

Nasal or other mucosal delivery of the therapeutic agent is alsocontemplated. Nasal delivery allows the passage to the blood streamdirectly after administering the composition to the nose, without thenecessity for deposition of the product in the lung. Formulations fornasal delivery include those with dextran or cyclodextran and saponin asan adjuvant.

The composition or vaccine of the present invention may be administeredin conjunction with one or more additional active ingredients,pharmaceutical compositions, or vaccines. The therapeutic agents of thepresent invention may be administered to an animal, preferably a mammal,most preferably a human.

Dosages

Following methodologies which are well-established in the art, effectivedoses and toxicity of the compounds and compositions of the instantinvention, which performed well in in vitro tests, are then determinedin preclinical studies using small animal models (e.g., mice or rats) inwhich the Shigella antigens, polypeptide, pharmaceutical, or vaccinecompositions have been found to be therapeutically effective and inwhich these drugs can be administered by the same route proposed for thehuman clinical trials.

Formulations or dosage forms for use in the present invention need notcontain a therapeutically effective amount of the components disclosedhere because such therapeutically effective amounts can be achieved byadministering a plurality of such formulations or dosage forms.

For any pharmaceutical composition used in the methods of the invention,the therapeutically effective dose can be estimated initially fromanimal models. Dose-response curves derived from animal systems are thenused to determine testing doses for the initial clinical studies inhumans. In safety determinations for each composition, the dose andfrequency of administration should meet or exceed those anticipated foruse in the clinical trial.

As disclosed herein, the dose of the components in the compositions ofthe present invention is determined to ensure that the dose administeredcontinuously or intermittently will not exceed an amount determinedafter consideration of the results in test animals and the individualconditions of a patient. A specific dose naturally varies depending onthe dosage procedure, the conditions of a patient or a subject animalsuch as age, body weight, sex, sensitivity, feed, dosage period, drugsused in combination, and seriousness of the disease. The appropriatedose and dosage times under certain conditions can be determined by thetest based on the above-described indices but may be refined andultimately decided according to the judgment of the practitioner andeach patient's circumstances (age, general condition, severity ofsymptoms, sex, etc.) according to standard clinical techniques.

Toxicity and therapeutic efficacy of the compositions of the inventioncan be determined by standard pharmaceutical procedures in experimentalanimals, e.g., by determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between therapeutic and toxic effects isthe therapeutic index and it can be expressed as the ratio ED₅₀/LD₅₀Compositions that exhibit large therapeutic indices are preferred.

The data obtained from animal studies can be used in formulating a rangeof doses for use in humans. The therapeutically effective doses of inhumans lay preferably within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage can vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. Ideally, a single dose of each drug should beused daily.

The following describes materials and methods employed in Examples 1-14.

Materials and Methods Cloning, Expression and Purification of ShigellaSigA2 and Icsp2 Polypeptides

DNA sequences of the protein antigens are available as described above(37). SigA DNA sequence of S. flexneri 2a strain 2457T was obtained fromthe gene bank accession number [Genbank AE014073]. The DNA sequence oficsP of S. flexneri 5a strain M90T was also obtained from the gene bankaccession number [Genbank AL391753]. The nucleotide identity and aminoacid sequence homology among different species of Shigella are more than99%. The DNA fragments were inserted into a commercially available E.coli over-expression plasmid pET21d (Novagen, Gibbstown, N.J., USA) andpurified according to the manufacturer's instruction using TALONmetal-affinity resin (Clontech, Mountain View, Calif., USA).

Primers:

IcsP2 primer set CCGGAATTCGGAGTGAAAACGGGGGGAGC (SEQ ID NO: 9) andCGGCGGCTCGAGCTAGTGGTGGTGGTGGTGGTGAATACTTGCACTATTTTT (SEQ ID NO: 10) wasused for amplification if IcsP2 fragment from S. flexneri 2a 2457Tstrain. The amplified fragment (SEQ ID NO:1, 390 bp, underlinedsequence) was digested with EcoRI and XhoI (boxed sequence, as shown inFIG. 12).

SigA2 primer set CCCGGGGAATTCGGGAAAAAGCCTTCAATAAAA (SEQ ID NO: 11) andCGGCGGCTCGAGCTAGTGGTGGTGGTGGTGGTGGTTGAAACTACTTTCGCCTG (SEQ ID NO: 12)was used for amplification of SigA2 fragment from S. flexneri 2a 2457Tstrain, The amplified fragment (SEQ ID NO:3, 795 bp, underlinedsequence) was digested with EcoRI and XhoI (boxed sequence, as shown inFIG. 13).

Amplified IcsP2 fragment and SigA2 fragments were inserted into EcoRIand XhoI site of pET21d and the recombinant DNA was verified withsequencing. The E. coli BL21 (DE3) (Novagen, Gibbstown, N.J., USA)bacteria were transformed with each recombinant plasmid and the proteinoverexpression was induced by 0.5 mM IPTG(Isopropyl-β-D-Thiogalactopyranoside) in the media at 37° C. E. coliBL21 (DE3) overexpressing each fragment was harvested and disrupted byfreeze/thaw followed by sonication in the presence of 6 M urea. The E.coli extract was centrifuged by 12,000×g and the supernatant was loadedon the pre-equilibrated (with 1× binding buffer: 20 mM TrisCl pH 7.9,500 mM NaCl 5 mM immidazole and 6 M urea) TALON resin column (3 ml). Thecolumn was washed with 20 ml of 1× binding buffer and 30 ml of 1×washing buffer (20 mM TrisCl pH 7.9, 500 mM NaCl, 15 mM imidazole and 6M urea) and the protein was eluted by 1× elution buffer (20 mM TrisCl pH7.9, 500 mM NaCl, 250 mM imidazole).

One other fragment of IcsP was also subcloned as described above (Primerset CCCGGGGAATTCACCACTAACTATCCACTTTT (SEQ ID NO: 13) andCGGCGGCTCGAGCTAGTGGTGGTGGTGGTGGTGACTGTAACGACTCTCTTGGT A) (SEQ ID NO: 14)

icsP and sigA gene disrupted mutant strains (S. flexneri 2a 2457Tbackground) construction: icsP and sigA gene disrupted mutant strainswere constructed individually by using an allele exchange method (17).Briefly, the internal 300 nt DNA fragments of icsP and sigA (fromnucleotide 61 to 360) were amplified with primers Ics5Tr/Ics3Tr (5′-GGCTCT AGA ACCACTAACTATCCACTT-3′ (SEQ ID NO: 15)/5′-GCC GAA TTC CCA GCT CTGGTC GGT CCA-3′ (SEQ ID NO: 16)) for icsA fragment and Sig5Tr/Sig3Tr(5′-GGC TCT AGA GAA CTG ACC CGG AAA GTT AGT-3′ (SEQ ID NO: 17)/5′-GCCGAA TTC GTA CGC ACC TCC TAA TGA-3′ (SEQ ID NO: 18)) for sigA fragment.These fragments were inserted into a suicide plasmid pSW23.oriT. Therecombinant plasmid pSWicsPTr and pSEsigATr were used to transform E.coli strain BW19610(pir⁺Amp^(s) Cm^(r)). Each plasmid was purified fromBW19610, and used to transform E. coli SM10λpir (pir⁺Tra⁺Amp^(s)Cm^(r)). E. coli SM10λpir was conjugated with S. flexneri 2a 2457T andthe chloramphenicol resistant S. flexneri was isolated on a CongoRed/streptomycin/chloramphenicol plate. In each knockout strain, thegene on the virulence plasmid and the genome are split into twofragments: 5′ end fragment of 360 nucleotides and 3′ end fragment. Thisdisruption of each gene on the mutant strains was confirmed by PCR andsequencing. Each strain was used for challenging the immunized mice.

Three other fragments of SigA protein were also purified as describeabove using primer sets as: SigA1 fragment primer setCCCGGGGAATTCGGTATGGCGAAACAGCATTTGC (SEQ ID NO: 19) andCGGCGGCTCGAGCTAGTGGTGGTGGTGGTGGTGCTCTTGTTTTTTACCATCCA (SEQ ID NO: 20)(824 by fragment using EcoRI and XhoI), SigA3 fragment primer setCCGGGGAAGCTTGACCCCTACAGAAAATAATA (SEQ ID NO: 21) andCGGCGGCTCGAGCTAGTGGTGGTGGTGGTGGTGCTCGCCATTGGTGTCACGCA (SEQ ID NO: 22)(822 by fragment using HindIII and XhoI), and SigA4 fragment primer setCCCGGGGAATTCGGGATAAAAAACATGAGCTGG (SEQ ID NO: 23) andCGGCGGCTCGAGCTAGTGGTGGTGGTGGTGGTGGAAAGAGTAACGGAAGTTG G (SEQ ID NO: 24)(702 by fragment using EcoRI and XhoI). These fragments wereoverexpressed and purified as described above. The SigA1 fragment showedimmunogenicity but no protection while SigA3 and SigA4 fragments did notprovoke antibody responses in mice. All the primers were purchased fromGenotech, Taejon, Korea.

Bacterial Strains

S. flexneri 2a strain 2457T and S. flexneri 5a strain M90T were providedby Dr. Philippe Sansonetti, Institut Pasteur, Paris, France).

S. boydii (IB8295) serotype 1: was obtained from a Shigellosis patientin Pakistan collected in 2002 (IVI collection).

S. sonnei (1B4200): was obtained from a Shigellosis patient in Indiacollected in 2004 provided by Dr. G.B. Nair (NICED, India).

S. dysenteriae serotype 1: was provide by Dr. D. Kopecko (FDA, Bethesda,Md., USA).

Animal Immunizations

All animals were maintained under specific pathogen-free conditions inthe animal care facilities of the International Vaccine Institute(Seoul) in accordance with International guidelines and all experimentsdescribed here were approved by the International Vaccine Instituteethical committees for animal experimentation.

For the purpose of this invention, an immunologic adjuvant is defined as“any substance that acts to accelerate, prolong, or enhanceantigen-specific immune responses when used in combination with specificvaccine antigens” (The Use of Conventional Immunologic Adjuvants in DNAVaccine Preparations, by Shin Sasaki and Kenji Okuda. In D. B. Lowrieand R. G. Whalen (editors), DNA Vaccines: Methods and Protocols, HumanaPress, 2000. ISBN 978-0-89603-580-5), as well as “a substance used tohelp boost the immune response to a vaccine so that less vaccine isneeded (Definition of Adjuvant, National Cancer Institute;www.cancer.gov/templates/db_alpha.aspx? CdrID=43987). Cholera toxin,oligodeoxynucleotides which contain unmethylated CpG motifs (CpG ODN),and aluminium salts are known adjuvants, when co-administered withantigens.

Five to six week old female Balb/c mice were immunized with the proteinantigens administered by various routes. Dose: for each immunization, adose of 25 μg of the protein antigens was mixed with an adjuvant inisotonic, pyrogen-free, phosphate-buffered saline, pH 7.4 (PBS). Theadjuvant consisted of either cholera toxin (CT) (3 to 5 micrograms μg)per dose), CpG ODN (4 μg per dose), or aluminium hydroxide (alum).

Intra-Peritoneal Administration

25 μg of protein antigen was administered by intraperitoneal injection,with or without adjuvant, in a total volume of 0.2 ml of PBS. Mice wereimmunized three times, at two weeks intervals.

Intranasal Administration

25 μg of protein antigen was mixed with 3 μg of cholera toxin (CT) andadministered through each nostril in a total volume of 50 microliters(approximately 25 microliter per nostril). Two and four weeks later,mice were re-immunized under identical conditions.

Rectal Administration

100 μg protein antigen was mixed with CT (5 microgram) and administeredin a final volume of 0.2 milliliters with a pipette inserted into theanorectal orifice. Guinea pigs were immunized three times, at two weeksinterval between each immunization.

Animal Models of Shigellosis

Mouse pneumonia model: 2×10⁷ CFU of S. flexenri 2a strain 2457T in 50microliters of PBS were inoculated into the nostrils of immunized mice.Animals were monitored daily for 10 days. S. flexneri 5a strain M90T andS. dysenteriae 1 strains were also used for challenge experiment.

Guinea pig keratoconjunctivitis model: One week after the last of threeconsecutive immunizations with Shigella protein antigens adjuvanted withCT, guinea pigs were anesthetized and 1×10⁵ cfu/20u1 of S. flexneri 2astrain 2457T was instillated in the eye conjunctival sac of guinea pigs.Periocular symptoms were monitored daily for 4-5 days. For comparisonwith systemic immunization, 50 μg of protein was administered throughIntraperitoneal route with 3 μg of CT three times two weeks interval,and the immunized Guinea pig was challenged one week after the finalimmunization as described.

Guinea pig colitis model: A recently developed model of intestinalshigellosis (30) was used to evaluate the protective efficacy of thenovel Shigella common protein antigens disclosed in the presentinvention. Briefly, S. flexneri 2a 2457T (1×10⁹ CFU) or S. flexneri 5astrain M90T were administered by instillation through the ano-rectalorifice of 5 week old guinea pigs, as described by Shim, Suzuki, Changet al (30). Animals were examined daily for symptoms of dysentery(diarrhea, tenesmus) and then sacrificed by pentobarbital overdose priorto histological analyses of colon tissue specimens.

Measurements of Systemic and Mucosal Immune Responses to Shigella CommonProtein Antigens Collection of Biological Fluids

Serum and secretions (saliva, vaginal and rectal washes) were collected1 week before the first immunization and thereafter one week after eachimmunization.

Preparation of Organ Extracts and Isolation of Cell Suspensions

One week after the last immunization, mice were anesthetized withpentobarbital and 125 i.u. of heparin (SIGMA, MO, USA) in 0.2 ml salinewas injected intra-peritoneally. Blood was drawn directly from the heartand the mice were sacrificed by cervical dislocation. Mice were perfusedby injecting 15 ml of PBS containing heparin (10 i.u./ml) into the heartright ventricle until the lungs were inflated and turned clear. Finelycut lung fragments were digested for 30 min at 37° C. with collagenase A(0.5 mg/ml) (Roche) in RPMI medium (Gibco Europe, U.K.) supplementedwith DNase 1 (0.1 mg/ml) (Roche) and single cell suspensions werecollected by filtration through a cell strainer. Single cell suspensionsfrom spleen were obtained by pressing the organs through nylon sieves.All suspensions were freed from erythrocytes by treatment with ammoniumchloride, washed, resuspended in RPMI medium containing 5% FBS, andstored on ice until being assayed (within 30 minutes) by means of theELISPOT assay described below.

For preparation of cell-free organ extracts, the PERFEXT technique (34)was used. Lungs from perfused animals were excised and sliced into small(2-3 mm thick) fragments and further perfused by incubation for 30minutes at 37° C. in PBS containing heparin, under constant agitation.Fragments were pelleted by centrifugation (500 rpm, 3 minutes) andresuspended in extraction buffer, consisting of Triton X100, PMSF andprotease inhibitors (34). Samples were snap frozen in liquid nitrogenand stored at −70° C. Prior to use, samples were thawed at roomtemperature and centrifuged (2000 rpm, 5 minutes). Supernatants werecollected and assayed for specific antibody activity by means of ELISA,as described below.

Antibody ELISA (Enzyme Linked Immunosorbent Assay)

The levels of antibodies to Shigella common protein antigens in sera, insecretions and in tissue PERFEXT extracts, were estimated by standardsolid phase enzyme-linked immunosorbent assay (ELISA). Individual wellsof polystyrene 96-well plates (NUNC, Denmark) were coated with eachprotein (1 microgram per ml of PBS; 0.1 ml per well; overnightincubation at ambient temperature), blocked with 5% (vol/vol) skim milkin PBS containing 0.05% Tween 20 (0.2 ml per well; 30 minutes at ambienttemperature) and washed three times with PBS-Tween. Serial two-folddilutions of samples in PBS-Tween with 5% skim milk solution wereincubated in antigen-coated wells for 2 hrs at ambient temperature, andthe plates were washed three times with PBS-Tween to remove unboundantibodies. Next, 0.1 ml of PBS-Tween containing appropriately diluted(1/5000) horseradish peroxidase-conjugated goat anti-mouse IgA or IgGantibodies (Southern Biotechnology, Birmingham, Ala., USA) was added toindividual wells. The plates were then washed three times with PBS andenzyme-bound activity was monitored after addition of a chromogenicenzyme substrate. Color development was stopped by adding 50 μl of 0.5NH₂SO₄, and measured spectrophotometrically (O.D₄₅₀) using an ELISAreader. Data are expressed as geometric mean antibody titers, a titerbeing defined as the reciprocal of the highest dilution of a sampleyielding an absorbance value equal or above that of control (no sample)added.

ELISPOT assay: The frequency of cells producing specific antibodies toShigella protein antigens was determined by means of the enzyme-limkedimmunospot (ELISPOT) assay (4). For comparison, the frequency of cellsproducing antibodies to cholera toxin (CT) was determined whenapplicable. Briefly, 10 μg of cholera toxin, 50 μg of sigA2, and 100 μgof sigA2 protein diluted in PBS were added to individual wells ofnitrocellulose-bottomed 96 wells HA plates (Millipore, Bedford, USA).After overnight incubation at 4° C., each well was washed with PBS andblocked with RPMI culture medium (GIBCO, UK) containing 10% fetal bovineserum (FBS). Lung and spleen cell suspensions from immunized mice wereincubated in serial two-fold dilutions (starting at eight hundredthousand mononuclear cells per well) in RPMI medium with FBS. After a 4hrs incubation at 37° C., individual wells were washed 5 times with PBS,5 times with PBS containing 0.05% Tween 20 and then exposed for 1 hourat room temperature to 0.1 ml of PBS-Tween containing horseradishperoxidase conjugated goat antibodies to mouse IgG or mouse IgA (1:1000dilution). After 3 washes with PBS-Tween and 4 washes with PBS, wellswere exposed to chromogen substrate for 10-20 minutes until spotsappeared. Plates were then washes with running tap water and dried.Spots were then enumerated using a stereomicroscope. Data were expressedas numbers of spot-forming cells (SFC) adjusted to one million cells.

Example 1 Mucosal Immunization with IcsP2 Protects Mice AgainstPneumonia Induced by Shigella flexneri 2a

Mice were immunized with IcsP2 given together with CT by the intranasalroute, as described above. Animals were challenged with a lethal dose S.flexneri 2a strain 2457T. The results of FIG. 1 show that immunizationwith IcsP protein protects animals against lung challenge with S.flexneri 2a. From the results shown in FIG. 1, intranasal administrationof a live-attenuated S. flexneri vaccine strain (SC602) also protectedmice against challenge.

Example 2 Mucosal Immunization with Icsp2 Protects Mice AgainstPneumonia Induced By Distinct Serotypes of Shigella flexneri

Mice were immunized with IcsP2 given together with CT by the intranasalroute as described above. Animals were challenged with a lethal dose ofS. flexneri 5a strain M90T (2×10⁷ CFU in 50 μl). The results shown inFIG. 2 indicate that immunization with IcsP protein protects animalsagainst lung challenge with S. flexneri 5a. In contrast, intranasaladministration of live-attenuated S. flexneri 2a strain (SC602) failedto protect mice against a strain of Shigella belonging to a different(5a) serotype (as shown by the results summarized with the filledsquares in FIG. 2).

Example 3 Mucosal Immunization with Icsp2 Protects Mice AgainstPneumonia Induced by Shigella dysenteriae

Mice were immunized with IcsP2 given together with CT by the intranasalroute as described above. Animals were challenged with a lethal dose S.dysenteriae type 1 (strain provided by Dr. D. Kopecko, FDA, Bethesda,Md., USA).

The results presented in FIG. 3 demonstrate that when animals had beenimmunized with IcsP2, they were protected against challenge by anotherShigella species, namely Shigella dysenteriae type 1. In contrast,SC602, a live-attenuated strain of S. flexneri 2a, failed to protectmice against lethal lung challenge with S. dysenteriae type 1.

From the above experiments, it can be concluded that IcsP2 not onlyprotect sagainst mucosal infection with S. flexneri belonging todistinct serotypes but also against an infection caused by a differentspecies of Shigella, such as S. dysenteriae.

Example 4 Mucosal Immunization with SigA2 Protects Against S. flexneri2a (2457T) Challenge but not Against S. flexneri 5a (M90T)

SigA protein is known to be present exclusively in S. flexneri 2a andnot other serotypes of S. flexneri (1). Mice immunized with SigA2protein were challenged with lethal dose of S. flexneri 2a (2457T,filled circle) and S. flexneri 5a (M90T, filled square). Mice challengedwith 2457T showed 80% survival while the mice challenged with S.flexneri died by bacteria-induced pneumonia. Mice that were immunizedwith PBS showed 100% death by two strains 2457T (open circle in FIG. 4)and M90T (open square in FIG. 4).

Example 5 Immunization with IcsP2 Protects Against ExperimentalKeratoconjunctivitis

Guinea pigs immunized with IcsP2 and control (sham immunized with PBS)as described above. Animals were challenged one week after the last of 3consecutive immunizations with IcsP2 and CT adjuvant administered by theintranasal or the intraperitoneal route with 1×10⁵ colony-forming unitsof virulent S. flexneri 2a 2457T. Guinea pigs were then examined at 24hrs and 48 hrs after challenge for signs and intensity of ocularinflammation (keratoconjunctivitis). Animals with no detectableinflammation were considered protected. As can be seen in the Table 1below, 40 to 50% of animals immunized with IcsP2 administered by theintranasal or by the intraperitoneal route were protected againstShigella-induced keratoconjunctivitis, whereas control animals (treatedwith PBS) all displayed keratoconjunctivitis.

TABLE 1 antigen Intra-peritoneal immunization Intra-nasal immunizationIcsP2 2/4 (50%)* 2/5 (40%) PBS 4/4 (0%) 4/4 (0%) *Protective efficacy:percentage of animals protected

Example 6 Immunization with IcsP2 SigA2 Protects AgainstShigella-Induced Experimental Recto-Colitis

Guinea pigs immunized with IcsP2, SigA2, SC602 and PBS were challengedwith virulent S. flexneri 2a strain 2457T with inoculum of 1×10⁹ CFU and24 hr after the inoculation were examined for diarrhea, hemorrhage ofthe colon, and frequency of tenesmus. As can be seen in Table 2, allcontrol (PBS-treated) animals had hemorrhage of the recto-colonicmucosa, most of whom had diarrhea (lack of solid faeces) and presentedwith signs of straining at stool, also called rectal tenesmus. Incontrast, animals immunized with AigA2 had no signs of rectocolitis butdisplayed tenesmus. Animals that had been immunized with live attenuatedS. flexneri 2a strain SC602 were protected against challenge withvirulent S. flexneri 2a (strain 2457T). Most importantly, guinea pigsthat had been immunized with purified IcsP2 were also fully protectedagainst challenge with S. flexneri 2a (strain 2457T).

TABLE 2 Mean frequency Animal Hemorrhagic of tenesmus Group no.rectocolitis diarrhea (per hour) IcsP2 0 − − 2 1 − − 2 − − 3 − − SigA2 0− − 12 1 − − 2 − − SC602 0 − − 2 1 − − 2 − − 3 − − PBS 0 + + 15 1 + −2 + + 3 + +

Example 7 IcsP2 Induces Protective Immunity in Shigella-Induced GuineaPig Colitis Model with Different Serotypes of S. flexneri and S.dysenteriae Type 1.

TABLE 3 Challenge Strain Protection against colitis S. flexneri 2a 100%S. flexneri 5a 100% S. dysenteriae 1  80% SC602 (intrarectal) 100%

Example 8 Mucosal Administration of SigA2 or IcsP2 Given Together withCT Adjuvant Induces Serum Antibody Responses.

Serum antibody levels were then determined one week after the thirdintranasal immunization. As can be seen in FIG. 5, mice immunized witheither Shigella protein in the amount of 10 μg of each protein, for eachimmunization, mounted vigorous serum IgG antibody responses to thecorresponding antigen, which were manifest already after the secondimmunization, being further enlarged by a third vaccination. FIG. 5shows the antibody titers after each of 3 consecutive immunizations areshown for SigA2 and IcsP2 proteins.

Example 9 Mucosal Administration of Shigella Common Protein AntigensInduces Systemic and Mucosal Immune Responses

Antibody-secreting cells in spleen and lung of mice immunized with SigA2or IcsP2 were enumerated by ELISpot assay performed on cell suspensionscollected one week after 3 intra-nasal immunizations with SigA2 or IcsP2adjuvanted with CT. Results presented in FIG. 6A-B are expressed as meannumbers of ASCs per million mononuclear cells determined on groups of3-4 mice (histograms) plus standard error of the mean (vertical lines).As can be seen, animals immunized with SigA2 mounted predominantly IgA-and also IgG-ASC responses in both spleen (FIG. 6A) and lungs (FIG. 6B).Very similar findings were obtained for IcsP2-specific IgA- and IgG-ASCresponses in mice immunized with IcsP2.

Lung extracts from mice immunized with SigA2 by the intra-nasal (i.n.)route or the intra-peritoneal (i.p.) route were assayed forSigA2-specific IgG antibody activity one week after the last of 3consecutive immunizations. As can be seen in FIG. 7, systemic (i.p.) aswell as mucosal (i.n.) immunization with SigA2 induced antibodyresponses in the lungs and in serum. Similar lung responses wererecorded in mice immunized with IcsP2 after intranasal administration ofIcsP2.

Taken together, these results demonstrate that mucosally administeredSigA2 and IcsP2 are immunogenic and can induce systemic and mucosalantibody responses.

Example 10 Serum Antibodies to IcsP2 and SigA2 React with Full-LengthIcsP and SigA Shigella Proteins

Western blot analyses of Shigella proteins: SDS-PAGE was performed onwhole cell detergent extracts of S. flexneri serotype 2a 2457T, S.boydii, S. sonnei and the proteins were transferred on nitrocellulosemembranes. Membranes were incubated with mouse antisera diluted to 1/50or 1/100 (in PBS-Tween 20 for 2 hrs at ambient temperature. Theseantisera were obtained by intraperitoneal injections of SigA2 and IcsP2co-administered with alum adjuvant, as described above. After washingwith PBS-Tween, the membranes were further incubated with alkalinephosphatase-conjugated goat antibodies to mouse Ig (Southern Biotech,Birmingham, Ala., USA). After washing, membranes were developed byadding BCIP-NBT chromogen substrate. For comparative purposes, separatemembranes were stained with Coomassie Blue. As can be seen in FIGS. 9Aand 9B, antiserum raised against SigA2 and IcsP2 can recognize fulllength SigA and IcsP2 (arrow corresponding to the position of theproteins) of different Shigella species (S. flexneri 2a, S. boydii, S.sonnei, and S. dysenteriae).

Example 11 In Vitro Inhibitory Effects of Antisera to SigA2

Confluent HeLa cells were infected with an invasive S. flexneri 2a 2457Tstrain at a multiplicity of infection (m.o.i.) of 100, 10, and 1 for 2hours at 37° C. The bacteria were washed and cultures were overlaid withagarose. Plaques were visualized by Giemsa staining, performed 48 hoursafter the infection. To test antisera to SigA2 for its capacity toinhibit plaque formation, invasive bacteria and test antiserum weremixed for 20 min prior to being added to the cells (FIGS. 9A-B). Plaquereduction was monitored after Giemsa staining. As can be seen in FIG.9B, antibodies to SigA2 inhibited plaque formation induced by S.flexneri. At M.O.I 10, the number of plaques induced by invasive S.flexneri 2a in infected cells was reduced by about 60% reduction in thepresence of mouse antiserum to SigA2 (FIG. 9B) compare to controlwithout antiserum (FIG. 9A).

Example 12 Confirmation of the Presence of IcsP and SigA in ShigellaStrains

The presence of IcsP and SigA in Shigella strains was confirmed by PCRusing the primers used for construction of overexpression vectorsdescribed in the above section entitled “Cloning, expression andpurification of Shigella SigA2 and Icsp2 polypeptides.”

The presence of sigA gene and icsP gene in each serotype of Shigellaspp. was confirmed by PCR with primer sets for SigA2 and IcsP2fragments, as shown in FIG. 10. Nucleic acid fragments of sigA2 andicsP2 were amplified by PCR from all the strains except for sigA in S.dysenteriae, the sigA gene is known to be absent in S. dysenteriaestrains by whole genome sequencing results (39).

Example 13 Antibodies Against SigA2 Inhibit Keratoconjunctivitis by S.flexneri 2a

Virulent S. flexneri 2a strain 2457T (2×10⁴ colony-forming units in 20microliter) was mixed with PBS, the same volume of antiserum againstSigA2, or pre-immune sera, and then inoculated into the conjunctival sacof guinea pigs. As can be seen in FIG. 11A, the upper left panel showsmoderate (24 hrs) periocular inflammation in a control animal whichbecomes severe at 48 hrs (upper right panel, FIG. 11B) after inoculationof S. flexneri 2a (strain 2457T). In contrast, and as can be seen in thebottom two panels of FIG. 11, an antiserum to SIgA2 inhibited ocularinflammation induced by co-administered S. flexneri (strain 2457T)bacteria after 24 hours (FIG. 11C) and after 48 hours (FIG. 11D). Guineapig inoculated with bacteria mixed with pre-immune serum developedkeratoconjunctivitis.

Example 14 icsP and sigA Disrupted Mutant Strains can Escape fromImmunities Induced by IcsP2 and SigA2, Respectively

Mice were immunized with IcsP2, SigA2 or SC602 (a live-attenuated S.flexneri 2a strain), respectively. Animals were challenged with S.flexneri 2a or with S. flexneri 2a deleted of either IcsP or SigA(herein referred to as KO strains). As shown in Table 4, mice survive achallenge with S. flexneri 2a but succumbed to challenge with KOstrains, demonstrating the specificity of protection induced by IcsP2and SigA2 respectively.

TABLE 4 Survival of mice after challenge with virulent strains S.flexneri S. flexneri S. flexneri 2a icsP 2a sigA S. flexneri S.dysenteriae 2a KO strain KO strain 5a 1 S. boydii S. sonnei IcsP2  80% 0 70% 60% 60% 70% 80% SigA2 >80%  80% 0 0 0 80% 80% SC602 100% 100% 100%0 0 0 0 (S. flexneri 2a)

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The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims. It is further to be understood that allvalues are approximate, and are provided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1. A vaccine composition for immunizing a mammal against Shigellacomprising an amount of Shigella proteins IcsP2 or SigA2 proteinseffective to elicit an immune response against Shigella, and apharmaceutically acceptable carrier or diluent.
 2. A vaccine compositionfor immunizing a mammal against Shigella comprising an amount ofShigella protein IcsP2 effective to elicit an immune response againstShigella, and a pharmaceutically acceptable carrier or diluent.
 3. Thevaccine of claim 2 wherein said IcsP2 protein is chemically conjugatedto a carrier protein.
 4. A vaccine composition for immunizing a mammalagainst Shigella comprising an amount of Shigella SigA2 effective toelicit an immune response against Shigella, and a pharmaceuticallyacceptable carrier or diluent.
 5. The vaccine of claim 2 wherein saidIcsP2 protein is the product of genetic fusion of a polynucleotideencoding the sequences of IcsP2 and the sequence of a carrier protein.6. The vaccine of claim 5 wherein said SigA2 protein is chemicallyconjugated to a carrier protein.
 7. The vaccine of claim 5 wherein suchSigA2 protein is the product of a genetic form of a carrier and saidSigA2 proteins.
 8. The vaccine of claim 3, wherein the carrier proteinis selected from the group consisting of tetanus toxoid, diptheriatoxoid, cholera toxin B subunit, E. coli enterotoxin B subunit andflagellin.
 9. The vaccine composition of claim 1, further comprising anadjuvant.
 10. The vaccine composition of claim 5, further comprising anadjuvant wherein said adjuvant is an oil phase of an emulsion selectedfrom a group consisting of a water-in-oil emulsion and a double oilemulsion. 11-20. (canceled)
 21. A method of treating a mammal sufferingfrom or susceptible to a pathogenic infection caused by Shigella,comprising administering an effective amount of the vaccine compositionof claim
 1. 22. The method of claim 21, wherein the effective amount ofthe vaccine composition ranges between about 10 micrograms and about 2milligrams.
 23. A method for modulating the immune response of a mammalcomprising administering an effective amount of any one of the vaccinecomposition of claim
 1. 24. The method of claim 22, wherein theeffective amount of the vaccine composition ranges between about 10micrograms and about 2 milligrams. 25-34. (canceled)
 35. The vaccinecomposition of claim 2, further comprising an adjuvant.
 36. The vaccinecomposition of claim 4, further comprising an adjuvant.
 37. A method oftreating a mammal suffering from or susceptible to a pathogenicinfection caused by Shigella, comprising administering an effectiveamount of the vaccine composition of claim
 2. 38. A method of treating amammal suffering from or susceptible to a pathogenic infection caused byShigella, comprising administering an effective amount of the vaccinecomposition of claim 4.